Correction: Secretome profiling of PC3/nKR cells, a novel highly migrating prostate cancer subline derived from PC3 cells.

[This corrects the article DOI: 10.1371/journal.pone.0220807.].

Secretome profiling of PC3/nKR cells, a novel highly migrating prostate cancer subline derived from PC3 cells.

Prostate cancer (PCa) is the most common cancer among men worldwide. Most PCa cases are not fatal; however, the outlook is poor when PCa spreads to another organ. Bone is the target organ in about 80% of patients who experience metastasis from a primary PCa tumor. In the present study, we characterized the secretome of PC3/nKR cells, which are a new subline of PC3 cells that were originally isolated from nude mice that were implanted with PC3 cells without anti-natural killer (NK) cell treatment. Wound healing and Transwell assays revealed that PC3/nKR cells had increased migratory and invasive activities in addition to a higher resistance to NK cells-induced cytotoxicity as compared to PC3 cells. We quantitatively profiled the secreted proteins of PC3/nKR and PC3 cells by liquid chromatography-tandem mass spectrometry analysis coupled with 2-plex tandem mass tag labeling. In total, 598 secretory proteins were identified, and 561 proteins were quantified, among which 45 proteins were secreted more and 40 proteins were secreted less by PC3/nKR cells than by PC3 cells. For validation, the adapter molecule crk, serpin B3, and cystatin-M were analyzed by western blotting. PC3/nKR cells showed the selective secretion of NKG2D ligand 2, HLA-A, and IL-6, which may contribute to their NK cell-mediated cytotoxicity resistance, and had a high secretion of crk protein, which may contribute to their high migration and invasion properties. Based on our secretome analysis, we propose that PC3/nKR cells represent a new cell system for studying the metastasis and progression of PCa.

A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice.

Combination therapies with phosphoinositide 3-kinase (PI3K) inhibitors and trastuzumab (anti-human epidermal growth factor receptor [HER]2/neu antibody) are effective against HER2+ breast cancer. Isoform-selective PI3K inhibitors elicit anti-tumor immune responses that are distinct from those induced by inhibitors of class I PI3K isoforms (pan-PI3K inhibitors). The present study investigated the therapeutic effect and potential for stimulating anti-tumor immunity of combined therapy with an anti-HER2/neu antibody and pan-PI3K inhibitor (GDC-0941) or a PI3K p110α isoform-selective inhibitor (A66) in mouse models of breast cancer. The anti-neu antibody inhibited tumor growth and enhanced anti-tumor immunity in HER2/neu+ breast cancer TUBO models, whereas GDC-0941 or A66 alone did not. Anti-neu antibody and PI3K inhibitor synergistically promoted anti-tumor immunity by increasing functional T cell production. In the presence of the anti-neu antibody, A66 was more effective than GDC-0941 at increasing the fraction of CD4+, CD8+, and IFN-γ+CD8+ T cells in the tumor-infiltrating lymphocyte population. Detection of IFN-γ levels by enzyme-linked immunospot assay showed that the numbers of tumor-specific T cells against neu and non-neu tumor antigens were increased by combined PI3K inhibitor plus anti-neu antibody treatment, with A66 exhibiting more potent effects than GDC-0941. In a TUBO (neu+) and TUBO-P2J (neu-) mixed tumor model representing immunohistochemistry 2+ tumors, A66 suppressed tumor growth and prolonged survival to a greater extent than GDC-0941 when combined with anti-neu antibody. These results demonstrate that a PI3K p110α-isoform-selective inhibitor is an effective adjunct to trastuzumab in the treatment of HER2-positive breast cancer.

3,3'-Diindolylmethane inhibits VEGF expression through the HIF-1α and NF-κB pathways in human retinal pigment epithelial cells under chemical hypoxic conditions.

Oxidative stress in the retinal pigment epithelium (RPE) can lead to the pathological causes of age-related macular degeneration (AMD). Hypoxia induces oxidative damage in retinal pigment epithelial cells (RPE cells). In this study, we investigated the capacity of 3,3'-diindolylmethane (DIM) to reduce the expression of vascular endothelial growth factor (VEGF) under hypoxic conditions, as well as the molecular mechanisms involved. Human RPE cells (ARPE-19 cells) were treated with cobalt chloride (CoCl2, 200 µM) and/or DIM (10 and 20 µM). The production of VEGF was measured by enzyme-linked immunosorbent assay. The translocation of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) was determined by western blot analysis. The binding activity of HIF-1α and NF-κB was analyzed by electrophoretic mobility shift assay. The phosphorylation levels of mitogen-activated protein kinases (MAPKs) were measured by western blot analysis. The levels of mitochondrial reactive oxygen species (ROS) were detected by fluorescence microplate assay. The results revealed that DIM significantly attenuated the CoCl2-induced expression of VEGF in the ARPE-19 cells. The CoCl2-induced translocation and activation of HIF-1α and NF-κB were also attenuated by treatment with DIM. In addition, DIM inhibited the CoCl2-induced activation of p38 MAPK in the ARPE-19 cells. Pre-treatment with YCG063, a mitochondrial ROS inhibitor, led to the downregulation of the CoCl2-induced production of VEGF by suppressing HIF-1α and NF-κB activity. Taken together, the findings of our study demonstrate that DIM inhibits the CoCl2-induced production of VEGF by suppressing mitochondrial ROS production, thus attenuating the activation of HIF-1α and p38 MAPK/NF-κB.

Inhibitory effects of the platelet-activating factor receptor antagonists, CV-3988 and Ginkgolide B, on alkali burn-induced corneal neovascularization.

PURPOSE: Platelet-activating factor (PAF) has been found in various ocular tissues; the activity of PAF depends on the binding to its specific receptor, PAF-receptor. We investigated the therapeutic effects of PAF-receptor antagonists (CV-3988 and Ginkgolide B) on alkali burn-induced corneal neovascularization (CNV). METHODS: CNV was induced by applying a 0.2 N sodium hydroxide (3 µl, NaOH) solution directly on mice corneas. CV-3988 (1 mM/10 µl) and Ginkgolide B (1 mM/10 µl) were administered topically on the corneas three times daily for three consecutive days. CNV was evaluated under a slit-lamp microscope. Corneas were processed for histological, immunohistochemical and reverse transcription polymerase chain reaction analysis. Human umbilical vein endothelial cells were used for the migration and tube formation assay. RESULTS: Application of CV-3988 and Ginkgolide B inhibited CNV caused by alkali burn. CV-3988 and Ginkgolide B attenuated the expression of PAF-receptor mRNA. Alkali injury induced a massively increased intraocular mRNA expression of an angiogenic factor in cornea tissues, whereas these increments were attenuated by the application of CV-3988 and Ginkgolide B. CONCLUSIONS: CV-3988 and Ginkgolide B reversed opacity and neovascularization in alkali burn-induced corneas. Our findings suggest that CV-3988 and Ginkgolide B may be therapeutically useful in the treatment of CNV and inflammation.

Caffeic acid phenethyl ester inhibits the inflammatory effects of interleukin-1ß in human corneal fibroblasts.

CONTEXT: Expression of various inflammatory mediators in corneal fibroblasts contributes to corneal inflammation. OBJECTIVE: The purpose of this study was to assess the possible effects of caffeic acid phenethyl ester (CAPE) on the expression of inflammatory mediators during an inflammatory response in human corneal fibroblasts. MATERIALS AND METHODS: The levels of interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule-1 (ICAM-1) from IL-1ß-exposed human corneal fibroblasts were measured with enzyme-linked immunosorbent assays (ELISA). The regulatory mechanisms of CAPE on cellular signaling pathways were examined using Western blot and electrophoretic mobility shift assays. A functional validation was carried out by evaluating the inhibitory effects of CAPE on neutrophil and monocyte migration in vitro. RESULTS: CAPE inhibited the expression of IL-6, MCP-1 and ICAM-1 induced by the pro-inflammatory cytokine IL-1ß in corneal fibroblasts. The activation of AKT and NF-κB by IL-1ß was markedly inhibited by CAPE, whereas the activity of mitogen-activated protein kinases (MAPKs) was not affected. CAPE significantly suppressed the IL-1ß-induced migration of differentiated (d)HL-60 and THP-1 cells. DISCUSSION: These anti-inflammatory effects of CAPE may be expected to inhibit the infiltration of leukocytes into the corneal stroma in vivo.

Effects of novel isoform-selective phosphoinositide 3-kinase inhibitors on natural killer cell function.

Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110α has received considerable attention in oncology because the gene encoding p110α (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110α in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110α and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110α-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110α inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-γ and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110δinhibitor, with lesser effects of p110α inhibitors. Oral administration of mice with MLN1117, a p110α inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110α inhibitors to preserve NK cell function.

CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 µl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 µl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

Achieving cancer cell death with PI3K/mTOR-targeted therapies.

Inhibitors of the PI3K/mTOR signaling network are under development as novel cancer therapies. However, these compounds do not cause robust cytotoxic responses in tumor cells unless combined with other agents. Rational combinations with other targeted therapies will likely be necessary to achieve the potential of PI3K/mTOR inhibitors in oncology.

Selective inhibition of phosphoinositide 3-kinase p110α preserves lymphocyte function.

Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.

Expression of galectin-9 by IFN-γ stimulated human nasal polyp fibroblasts through MAPK, PI3K, and JAK/STAT signaling pathways.

Galectin-9 exhibited potent and selective eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo. Nasal polyposis is a chronic inflammatory disease of the upper airway characterized by the marked presence of inflammatory cells, particularly eosinophils. Thus, galectin-9 may be implicated in the pathogenesis of nasal polyposis. The study was designed to investigate whether interferon-gamma (IFN-γ) can induce the augmentation of galectin-9 expression and induce the expression of galectin-9 in nasal polyps. We examined the correlation between galectin-9 expression and eosinophil infiltration in nasal polyps. In addition, we identified the signaling pathways involved in the elevation of galectin-9 expression in response to IFN-γ. Our data demonstrate that the involvement of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3 phosphate kinase (PI3K), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) may play important roles in the selective recruitment of eosinophils in nasal polyp tissues through the production of galectin-9. These findings suggest that galectin-9 expression is associated with eosinophil infiltration in polyps of patients with nasal polyposis.

Cilostazol protects mice against endotoxin shock and attenuates LPS-induced cytokine expression in RAW 264.7 macrophages via MAPK inhibition and NF-kappaB inactivation: not involved in cAMP mechanisms.

Cilostazol, a phosphodiesterase 3 inhibitor, is a platelet aggregation inhibitor and vasodilator that is useful for treating intermittent claudication. Experimental studies have shown that cilostazol has potent anti-inflammatory effects. In the present study, we examined the effect of cilostazol on lipopolysaccharide (LPS)-induced inflammatory cytokines in macrophages and endotoxin shock in mice. Our results indicate that cilostazol inhibits LPS-stimulated up-regulation of pro-inflammatory cytokines in a concentration-dependent manner without appreciable cytotoxicity in RAW 264.7 cells. Cilostazol did not enhance intracellular cyclic AMP (cAMP) levels. To further elucidate the mechanism responsible for the inhibition of production of pro-inflammatory mediators by cilostazol, we examined the effect of cilostazol on LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation and phosphorylation of mitogen-activated protein kinases (MAPK). Our results clearly indicated that cilostazol treatment reduced on of MAPK phosphorylation and NF-kappaB activity, and that the inhibitory effect of cilostazol is independent of the cAMP pathway. In an animal model, cilostazol protected c57BL/6 mice from LPS-induced endotoxin shock, possibly through inhibition of the production of pro-inflammatory cytokines. In conclusion, cilostazol inhibits LPS-stimulated production of pro-inflammatory cytokines and protects mice from endotoxin shock, suggesting that cilostazol may be a novel therapeutic agent for the prevention of various inflammatory diseases.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: We examined the effects of cilostazol on LPS-induced nuclear factor-kappaB (NF-kappaB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs). KEY RESULTS: 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. CONCLUSION AND IMPLICATIONS: 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.

Magnolol Inhibits LPS-induced NF-κB/Rel Activation by Blocking p38 Kinase in Murine Macrophages.

This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-κB/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-κB/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-κB/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-κB/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-κB/Rel and p38 kinase signaling.

Constituents of Corydalis heterocarpa and their anti-proliferative effects on human cancer cells.

Two new coumarins, 1 and 2, along with four known coumarins (3-6) have been isolated from Corydalis heterocarpa. On the basis of spectroscopic and chemical methods, compounds 1 and 2 were elucidated as (2'S,7'S)-O-2-methylbutanoyl-columbianetin and (2'S)-columbianetin-3'-sulfate, respectively. The anti-proliferative activity against human cancer cells of compounds 1-6 isolated from C. heterocarpa was evaluated using a MTT assay and by mRNA expression of several factors related to apoptosis. Among them, compound 2 exerted the more potent anti-proliferative activity compared with the other compounds treated. The potent inhibitory effect of compound 2 was produced by induction of apoptosis through activating Bax, p53 and p21 expressions.

Evaluation of Salicornia herbacea as a potential antioxidant and anti-inflammatory agent.

In this study, the antioxidant and anti-inflammatory activities of Salicornia herbacea were evaluated. The crude CH(2)Cl(2)/methanol extract of S. herbacea showed 52% and 86% scavenging activities of the authentic ONOO(-) and ONOO(-) from 3-morpholinosydnomimine (SIN-1) at a concentration of 50 microg/mL, respectively, and was subjected to a further fractionation with n-hexane, 85% aqueous methanol, n-butanol, and water. Additional purification of the n-butanol fraction revealed that the most potent scavenging activity led to the isolation of isorhamnetin 3-O-beta-d-glucopyranoside as the active principle. The structure of isorhamnetin 3-O-beta-d-glucopyranoside was elucidated by extensive two-dimensional nuclear magnetic resonance experiments such as (1)H correlation spectroscopy nuclear Overhauser effect spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple-bond correlation as well as by comparison with the published spectral data. Isorhamnetin 3-O-beta-d-glucopyranoside exhibited dose-dependent scavenging activities of the authentic ONOO(-) and ONOO(-) from SIN-1. The electron spin resonance spin-trap techniques confirmed that reactive oxygen species, including the hydroxyl, superoxide, carbon-centered, and 1,1-diphenyl-2-picrylhydrazyl radicals, were actively quenched by addition of isorhamnetin 3-O-beta-d-glucopyranoside. In addition, isorhamnetin 3-O-beta-d-glucopyranoside suppressed the lipopolysaccharide-induced nitric oxide production and the expression of cytokines such as inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1beta in Raw 264.7 cells. Findings from this study should underscore the nutraceutical value of S. herbacea-derived isorhamnetin 3-O-beta-d-glucopyranoside as a potent antioxidative and anti-inflammatory agent via alleviation of radical-induced toxicities and pro-inflammatory responses.

The 15-deoxy-Delta12,14-prostaglandin J2 inhibits LPS-stimulated AKT and NF-kappaB activation and suppresses interleukin-6 in osteoblast-like cells MC3T3E-1.

AIMS: Periodontitis is a chronic inflammatory disease that results in gingival inflammation and periodontal tissue destruction and is accompanied by alveolar bone resorption and eventual tooth loss. We examined the effect of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) on periodontitis by inhibiting the production of interleukin-6 (IL-6). MAIN METHODS: Osteoblast-like cells MC3T3E-1 were pretreated with 15d-PGJ(2) before being incubated with lipopolysaccharide (LPS), the effect of 15d-PGJ(2) on IL-6 production, expression and its regulatory mechanisms were studied by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, electrophoretic mobility shift assay (EMSA), and confocal laser scanning microscopy study. KEY FINDINGS: 15d-PGJ(2) inhibits LPS-stimulated IL-6 production in a concentration-dependent manner in osteoblast-like cells MC3T3E-1, without appreciable cytotoxicity. To further examine the mechanism responsible for the inhibition of IL-6 production by 15d-PGJ(2), we examined the effect of 15d-PGJ(2) on nuclear factor-kappaB (NF-kappaB) activation and the phosphorylation of protein kinase B (Akt). 15d-PGJ(2) treatment clearly reduced the DNA binding activity of NF-kappaB in LPS-stimulated osteoblast-like cells MC3T3E-1, an effect that was mediated by inhibiting the degradation of inhibitor kappaB (IkappaB) and nuclear translocation of NF-kappaB p65 subunit. In addition, 15d-PGJ(2) attenuated the LPS-mediated Akt pathway. These effects of 15d-PGJ(2) were not abrogated by the PPARgamma antagonist, GW9662, indicating that they are PPARgamma-independent actions. SIGNIFICANCE: These results suggest that 15d-PGJ(2) possess a potent suppressive effect on inflammatory responses of osteoblast-like cells MC3T3E-1 via the Akt and NF-kappaB pathways independent of PPARgamma and suggest that this compound may offer some insight into the development of a new therapeutic approach to the prevention and treatment of periodontal diseases.

Ecklonia cava ethanolic extracts inhibit lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in BV2 microglia via the MAP kinase and NF-kappaB pathways.

Ecklonia cava (EC) is a brown alga that has demonstrated radical scavenging, bactericidal, tyrosinase inhibitory, and protease inhibitory activities. However, the molecular mechanisms underlying its anti-inflammatory action remain unclear. In the current study, we attempted to determine whether pretreatment with EC induces a significant inhibition of anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. Our results indicate that EC inhibits LPS-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in a concentration-dependent manner and inhibits inducible nitric oxide (iNOS) and cyclooxygenase (COX)-2 in BV2 microglia without significant cytotoxicity. EC treatment significantly reduced nuclear factor-kappaB (NF-kappaB) translocation and DNA-binding in LPS-stimulated BV2 microglia. This effect was mediated through the inhibition of the degradation of the inhibitor kappaB and by inhibition of the mitogen-activated protein kinase (MAPK) phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Our data also indicate that EC extracts exert anti-inflammatory effects by suppressing proinflammatory cytokines. Collectively, these results suggest that EC suppresses the induction of cytokines by LPS, as well as iNOS and COX-2 expression, by blocking NF-kappaB and MAPK activation. These findings provide mechanistic insights into the anti-inflammatory and neuroprotective actions of EC in BV2 microglia.

Amplification of an RNA ligase ribozyme under alternating temperature conditions.

Amplification of an RNA template molecule was examined using the ligase ribozyme and its corresponding RNA substrates under alternating temperature conditions. Alternating temperatures enhanced the rate of the thermodynamically unfavorable dissociation of the annealed products into the two separate RNA templates, reminiscent of the polymerase chain reaction. Under these conditions, the RNA ligase ribozyme system was observed to amplify through a mainly cross-catalytic process, generating additional copies of the starting RNA template molecules. Thus, template-directed RNA ligation using the ribozyme under thermally fluctuating conditions will be an intriguing point to consider when explaining the primordial event of chemical evolution.