6-(Methylsulfinyl)hexyl isothiocyanate suppresses inducible nitric oxide synthase expression through the inhibition of Janus kinase 2-mediated JNK pathway in lipopolysaccharide-activated murine macrophages

6-(Methylsulfinyl)hexyl isothiocyanate (6-MITC) is an active ingredient of Wasabi (Wasabia japonica (Miq.) Matsumura), which is a very popular pungent spice in Japan. To clarify the cellular signaling mechanism underlying the anti-inflammatory action of 6-MITC, we investigated the effects of 6-MITC on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. 6-MITC showed a dose-dependent inhibition of LPS-induced nitric oxide (NO), iNOS mRNA and protein. LPS caused the c-Jun phosphorylation (a major component of AP-1) and IkappaB-alpha degradation. 6-MITC suppressed LPS-induced c-Jun phosphorylation, but did not inhibit IkappaB-alpha degradation. Cellular signaling analysis using MAPK-(U0126 for MEK1/2, SB203580 for p38 kinase and SP600125 for JNK) and Jak2-specific (AG490) inhibitors demonstrated that LPS stimulated iNOS expression via activating Jak2-mediated JNK, but not ERK and p38, pathway. 6-MITC suppressed iNOS expression through the inhibition of Jak2-mediated JNK signaling cascade with the attendant to AP-1 activation. In addition, the structure-activity study revealed that the inhibitory potency of methylsulfinyl isothiocyanates (MITCs) depended on the methyl chain length. These findings provide the molecular basis for the first time that 6-MITC is an effective agent to attenuate iNOS production.     

Cryptotanshinone inhibits LPS-induced proinflammatory mediators via TLR4 and TAK1 signaling pathway

Cryptotanshinone (CTN), one of the major constituents of tanshinones, was investigated for anti-inflammatory activity in the murine macrophage cell line RAW 264.7. CTN inhibited the production of nitric oxide (NO) production, as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages. Since CTN was considered as inhibiting LPS-triggered phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation, we consequently evaluated the expression of toll-like receptor 4 (TLR4) and CD14, as well as phosphorylation of TGF-β-activated kinase 1 (TAK1). CTN reduced the expression of CD14 and TLR4, and suppressed LPS-induced phosphorylation of TAK1. Furthermore, CTN significantly increased the survival rate against LPS challenge in D-galactosamine-sensitized mice, which was in line with in vitro results. These results suggested that CD14/TLR4 and TAK1 might be the potential molecular targets for addressing the protective effects of CTN on LPS-induced inflammatory effects in macrophages.     

Protection against lipopolysaccharide-induced sepsis and inhibition of interleukin-1beta and prostaglandin E2 synthesis by silymarin

Silymarin is known to have hepatoprotective and anticarcinogenic effects. Recently, anti-inflammatory effect of silymarin is attracting an increasing attention, but the mechanism of this effect is not fully understood. Here, we report that silymarin protected mice against lipopolysaccharide (LPS)-induced sepsis. In this model of sepsis, silymarin improved the rate of survival of LPS-treated mice from 6 to 38%. To further investigate the mechanism responsible for anti-septic effect of silymarin, we examined the inhibitory effect of silymarin on interleukin-1beta (IL-1beta) and prostaglandin E2 (PGE2) production in macrophages. Silymarin dose-dependently suppressed the LPS-induced production of IL-1beta and PGE2 in isolated mouse peritoneal macrophages and RAW 264.7 cells. Consistent with these results, the mRNA expression of IL-1beta and cyclooxygenase-2 was also completely blocked by silymarin in LPS-stimulated RAW 264.7 cells. Moreover, the LPS-induced DNA binding activity of nuclear factor-kappaB/Rel was also inhibited by silymarin in RAW 264.7 cells. Taken together, these results demonstrate that silymarin has a protective effect against endotoxin-induced sepsis, and suggest that this is mediated, at least in part, by the inhibitory effect of silymarin on the production of IL-1beta and PGE2.     

Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase

We previously found that bee venom (BV) and melittin (a major component of BV) has anti-inflammatory effect by reacting with the sulfhydryl group of p50 of NF-kappaB. Since the sulfhydryl group is present in IkappaB kinase (IKKalpha and IKKbeta), anti-inflammatory effect of melittin via interaction with IKKs was investigated. We first examined binding of melittin to IKKs using surface plasmon resonance analyzer. Melittin binds to IKKalpha (K(d) = 1.34 x 10(-9) M) and IKKbeta (K(d) = 1.01 x 10(-9) M). Consistent with the high binding affinity, melittin (5 and 10 microg/ml) and BV (0.5, 1 and 5 microg/ml) suppressed sodium nitroprusside, TNF-alpha and LPS induced-IKKbeta and IKKbeta activities, IkappaB release, and NF-kappaB activity as well as the expressions of iNOS and COX-2, and the generation of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in Raw 264.7 mouse macrophages and synoviocytes obtained from rheumatoid arthritis patients. The binding affinities of melittin to mutant IKKs, was reduced, and the inhibitory effect of melittin on IKK and NF-kappaB activities, and NO and PGE(2) generation were abrogated by the reducing agents or in Raw 264.7 transfected with mutant plasmid IKKalpha (C178A) or IKKbeta (C179A). These results suggest that melittin binding to the sulfhydryl group of IKKs resulted in reduced IKK activities, IkappaB release, NF-kappaB activity and generation of inflammatory mediators, indicating that IKKs may be also anti-inflammatory targets of BV.     

Brazilin selectively disrupts proximal IL-1 receptor signaling complex formation by targeting an IKK-upstream signaling components

The ligation of interleukin-1 receptor (IL-1R) or tumor necrosis factor receptor 1 (TNFR1) induces the recruitment of adaptor proteins and their concomitant ubiquitination to the proximal receptor signaling complex, respectively. Such are upstream signaling events of IKK that play essential roles in NF-κB activation. Thus, the discovery of a substance that would modulate the recruitment of key proximal signaling elements at the upstream level of IKK has been impending in this field of study. Here, we propose that brazilin, an active compound of Caesalpinia sappan L. (Leguminosae), is a potent NF-κB inhibitor that selectively disrupts the formation of the upstream IL-1R signaling complex. Analysis of upstream signaling events revealed that brazilin markedly abolished the IL-1β-induced polyubiquitination of IRAK1 and its interaction with IKK-γ counterpart. Notably, pretreatment of brazilin drastically interfered the recruitment of the receptor-proximal signaling components including IRAK1/4 and TRAF6 onto MyD88 in IL-1R-triggerd NF-κB activation. Interestingly, brazilin did not affect the TNF-induced RIP1 ubiquitination and the recruitment of RIP1 and TRAF2 to TNFR1, suggesting that brazilin is effective in selectively suppressing the proximal signaling complex formation of IL-1R, but not that of TNFR1. Moreover, our findings suggest that such a disruption of IL-1R-proximal complex formation by brazilin is not mediated by affecting the heterodimerization of IL-1R and IL-1RAcP. Taken together, the results suggest that the anti-IKK activity of brazilin is induced by targeting IKK upstream signaling components and subsequently disrupting proximal IL-1 receptor signaling complex formation.     

Inhibition of inflammatory mediators by polyphenolic plant extracts in human intestinal Caco-2 cells

The mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) are involved in transduction cascades that play a key role in inflammatory response. We tested the ability of preselected natural polyphenolic extracts (grape seed, cocoa, sugar cane, oak, mangosteen and pomegranate) to modulate intestinal inflammation using human intestinal Caco-2 cells treated for 4 h with these extracts and then stimulated by cytokines for 24 or 48 h. The effect of polyphenolic extracts, at 50 μmol of gallic acid equivalent/l, was investigated on inflammation-related cellular events: (i) NF-κB activity (cells transfected with a NF-κB-luciferase construct), (ii) activation of Erk1/2 and JNK (western blotting), (iii) secretion of interleukin 8 (IL-8) (ELISA), (iv) secretion of prostaglandin (PG) E₂ (ELISA), (v) production of NO (Griess method). Results show that: (i) sugar cane, oak and pomegranate extracts inhibited NF-κB activity (from 1.6 to 1.9-fold) (P < 0.001); (ii) pomegranate slightly inhibited Erk1/2 activation (1.3-fold) (P = 0.008); (iii) oak and pomegranate decreased NO synthesis by 1.5-fold (P < 0.001) and that of IL-8 by 10.3 and 6.7-fold respectively; (iv) pomegranate and cocoa decreased PGE₂ synthesis by 4.6 (P < 0.0001) and 2.2-fold (P = 0.001), respectively. We suggest that pomegranate extract could be particularly promising in dietary prevention of intestinal inflammation.     

Inhibition of NF-kappa B activation through targeting I kappa B kinase by celastrol, a quinone methide triterpenoid

Celastrol, a quinone methide triterpenoid, was isolated as an inhibitor of NF-kappaB from Celastrus orbiculatus. This compound dose-dependently inhibited a variety of stimuli-induced NF-kappa B-regulated gene expression and the DNA-binding of NF-kappa B in different cell lines without affecting DNA-binding activity of AP-1. Preincubation of celastrol completely blocked the LPS-, TNF-alpha-, or PMA-induced degradation and phosphorylation of I kappa B alpha. Importantly, celastrol inhibited IKK activity and the constitutively active IKK beta activity in a dose-dependent manner without either affecting the NF-kappa B activation induced by RelA over-expression or directly suppressing the DNA-binding of activated NF-kappa B. However, mutation of cysteine 179 in the activation loop of IKK beta abolished sensitivity towards to celastrol, suggesting that celastrol suppressed the NF-kappa B activation by targeting cysteine 179 in the IKK. To verify that celastrol is a NF-kappa B inhibitor, we investigated its effect on some NF-kappa B target genes expressions. Celastrol prevented not only LPS-induced mRNA expression of iNOS and TNF-alpha, but also TNF-alpha-induced Bfl-1/A1 expression, a prosurvival Bcl-2 homologue. Consistent with these results, celastrol significantly suppressed the production of NO and TNF-alpha in LPS-stimulated RAW264.7 cells, and increased the cytotoxicity of TNF-alpha in HT-1080 cells. We also demonstrated that celastrol showed anti-inflammatory and anti-tumor activities in animal models. Taken together, this study extends our understanding on the molecular mechanisms underlying the anti-inflammatory and anti-cancer activities of celastrol and celastrol-containing medicinal plant, which would be a valuable candidate for the intervention of NF-kappa B-dependent pathological conditions.     

Gallic acid inhibits migration and invasion in human osteosarcoma U-2 OS cells through suppressing the matrix metalloproteinase-2/-9, protein kinase B (PKB) and PKC signaling pathways

Advanced cancer is a multifactorial disease which complicates treatment if the cancer cells have metastasized calling for the targeting of multiple cellular pathways. Gallic acid (GA) is known to possess multiple pharmacological activity including antitumor effects. This study investigated the mechanisms for the anticancer properties of GA on migration and invasion of human osteosarcoma U-2 OS cells. The migration and invasion in U-2 OS cells were determined by a Boyden chamber transwell assay. The expression levels and activities of MMP-2 and MMP-9 were measured by Western blotting, real-time PCR and gelatin zymography assays. All examined proteins levels from Western blotting indicated that GA decreased the protein levels of GRB2, PI3K, AKT/PKB, PKC, p38, ERK1/2, JNK, NF-κB p65 in U-2 OS cells. GA also inhibited the activities of AKT, IKK and PKC by in vitro kinase assay. GA suppressed the migration and invasive ability of U-2 OS cells, and it decreased MMP-2 and MMP-9 protein and mRNA levels and secreted enzyme activities in vitro. These results suggest that potential signaling pathways of GA-inhibited migration and invasion in U-2 OS cells may be due to down-regulation of PKC, inhibition of mitogen-activated protein kinase (MAPK) and PI3K/AKT, resulting in inhibition of MMP-2 and MMP-9 expressions.     

A synthetic analog of alpha-galactosylceramide induces macrophage activation via the TLR4-signaling pathways

Alpha-galactosylceramide (alpha-GalCer), a bioactive glycolipid isolated from the marine sponge Agelas mauritianus, is a potent immunomodulator with therapeutic potential for the treatment of autoimmune diseases and cancer. The Toll-like receptor 4 (TLR4), one of the promising molecular targets for immune-modulating drugs, is commonly expressed in innate immune cells especially macrophages and dendritic cells. Currently, whether alpha-GalCer can activate TLR4 signaling pathways remains unreported. In this study, we examined the effects of alpha-GalCer and its various structural analogs, CCL-1 approximately 47, on TLR4 activation. We found that one alpha-GalCer analog (CCL-34), but not alpha-GalCer itself, strongly stimulated NF-kappaB activity in RAW 264.7 cells. CCL-34 activated NF-kappaB in a TLR4-dependent manner and stimulated TNF-alpha production in bone marrow cells of TLR4-functional C3H/HeN mice but not in those of TLR4-defective C3H/HeJ mice. Furthermore, CCL-34 treatment stimulated NF-kappaB activation and IL-8 production in a 293 cell line constitutively expressing human TLR4, MD-2 and CD14. Treatment of RAW 264.7 cells with CCL-34 also activated TLR4-downstream mitogen-activated protein kinases (ERK, JNK and p38), induced expression of TLR4-downstream genes (TNF-alpha, IL-6, IL-1beta and iNOS) and promoted production of cytokines characteristic of activated macrophages. CCL-34-treated RAW 264.7 cells acquired a distinct morphology similar to that of LPS-activated macrophages and exhibited higher phagocytotic activity. Moreover, treatment with a TLR4-neutalizing antibody inhibited the CCL-34-induced morphological alteration. In summary, we identify a novel synthetic compound CCL-34 that can activate macrophages via TLR4-dependent signaling pathways. Our results suggest that CCL-34 is an immune modulator and may serve as a potential drug lead for immunotherapy.     

Suppression of RelA/p65 transactivation activity by a lignoid manassantin isolated from Saururus chinensis

In our search for NF-kappaB inhibitors from natural resources, we have previously identified two structurally related dilignans, manassantin A and B as specific inhibitors of NF-kappaB activation from Saururus chinensis. However, their molecular mechanism of action remains unclear. We here demonstrate that manassantins A and B are potent inhibitors of NF-kappaB activation by the suppression of transciptional activity of RelA/p65 subunit of NF-kappaB. These compounds significantly inhibited the induced expression of NF-kappaB reporter gene by LPS or TNF-alpha in a dose-dependent manner. However, these compounds did not prevent the DNA-binding activity of NF-kappaB assessed by electrophoretic mobility shift assay as well as the induced-degradation of IkappaB-alpha protein by LPS or TNF-alpha. Further analysis revealed that manassantins A and B dose-dependently suppressed not only the induced NF-kappaB activation by overexpression of RelA/p65, but also transactivation activity of RelA/p65. Furthermore, treatment of cells with these compounds prevented the TNF-alpha-induced expression of anti-apoptotic NF-kappaB target genes Bfl-1/A1, a prosurvival Bcl-2 homologue, and resulted in sensitizing HT-1080 cells to TNF-alpha-induced cell death. Similarly, these compounds also suppressed the LPS-induced inducible nitric oxide synthase expression and nitric oxide production. Taken together, manassantins A and B could be valuable candidate for the intervention of NF-kappaB-dependent pathological condition such as inflammation and cancer.     

Involvement of protein kinase Cdelta and extracellular signal-regulated kinase-2 in the suppression of microglial inducible nitric oxide synthase expression by N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast)

Excess nitric oxide (NO) in the brain released by microglial cells contributes to neuronal damage in various pathologies of the central nervous system (CNS) including neurodegenerative diseases and multiple sclerosis. N-[3,4-Dimethoxycinnamoyl]-anthranilic acid (tranilast, TNL) is an anti-allergic compound which suppresses the activation of monocytes. We show that inducible nitric oxide synthase (iNOS) mRNA and protein expression and the release of NO from N9 microglial cells stimulated with the bacterial endotoxin lipopolysaccharide (LPS) are inhibited when the cells are exposed to TNL. TNL fails to modulate LPS-stimulated nuclear factor-kappaB (NF-kappaB) reporter gene activity and phosphorylation of inhibitory kappaB (IkappaB), indicating that NF-kappaB is not involved in the TNL-mediated suppression of LPS-induced iNOS expression. Moreover, TNL inhibits LPS-induced phosphorylation of extracellular signal-regulated kinase 2 (ERK-2). Finally, TNL abolishes translocation of protein kinase Cdelta (PKCdelta) to the nucleus and suppresses the phosphorylation of the PKCdelta substrate, myristoylated alanin-rich C kinase substrate (MARCKS). We conclude that the anti-allergic compound TNL suppresses microglial iNOS induction by LPS via inhibition of a signalling pathway involving PKCdelta and ERK-2.     

Influence of deoxynivalenol on NF-kappaB activation and IL-8 secretion in human intestinal Caco-2 cells

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. In human intestinal Caco-2 cells, DON activates the mitogen-activated protein kinases (MAPKs). We hypothesized a link between DON ingestion and intestinal inflammation, and used Caco-2 cells to assess the effects of DON, at plausible intestinal concentrations (250-10,000 ng/ml), on inflammatory mediators acting downstream the MAPKs cascade i.e. activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 (IL-8) secretion. In addition, Caco-2 cells were co-exposed to pro-inflammatory stimuli in order to mimic an inflamed intestinal epithelium. Dose-dependent increases in NF-kappaB activity and IL-8 secretion were observed, reaching 1.4- and 7.6-fold, respectively using DON at 10 microg/ml. Phosphorylation of inhibitor-kappaB (IkappaB) increased (1.6-fold) at DON levels <0.5 microg/ml. Exposure of Caco-2 cells to pro-inflammatory agents, i.e. 25 ng/ml interleukin-1beta, 100 ng/ml tumor necrosis factor-alpha or 10 microg/ml lipopolysaccharides, activated NF-kappaB and increased IL-8 secretion. Synergistic interactions between these stimuli and DON were observed. These data show that DON induces NF-kappaB activation and IL-8 secretion dose-dependently in Caco-2 cells, and this effect was accentuated upon pro-inflammatory stimulation, suggesting DON exposure could cause or exacerbate intestinal inflammation.     

Dimeric procyanidin B2 inhibits constitutively active NF-kappaB in Hodgkin's lymphoma cells independently of the presence of IkappaB mutations

Due to long-term toxicity of current Hodgkin's lymphoma (HL) treatment, the present challenge is to find new therapies that specifically target deregulated signaling cascades, including NF-kappaB, which are involved in Hodgkin (H) and Reed-Sternberg (RS) cell proliferation and resistance to apoptosis. We previously presented evidence that dimeric procyanidin B2 (B2) can interact with NF-kappaB proteins inhibiting the binding of NF-kappaB to DNA. Herein, we investigated if B2, acting at a late event in NF-kappaB signaling cascade, could be effective in inhibiting NF-kappaB in H-RS cells with different mechanisms of constitutive NF-kappaB activation. B2 caused a concentration-dependent inhibition of NF-kappaB-DNA binding to a similar extent (41-48% inhibition at 25 microM B2) in all the tested H-RS cell lines (L-428, KM-H2, L-540, L-1236 and HDML-2). This was associated with the inhibition of NF-kappaB-driven gene expression, including cytokines (IL-6, TNFalpha and RANTES) and anti-apoptotic proteins (Bcl-xL, Bcl-2, XIAP and cFLIP). The finding of similar amounts of RelA and p50 proteins in the nucleus, but decreased NF-kappaB-DNA binding, even in those H-RS cells characterized by mutations in the inhibitory IkappaB proteins, supports that B2 acts by preventing the binding of NF-kappaB to DNA. B2 did not inhibit AP-1 and STAT3 constitutive activation in H-RS cells, indicating that the moderate effects of B2 on cell viability are due to the complex signaling aberrations in HL. Thus, several signaling pathways should be targeted when designing therapeutics for HL. In this regard, the capacity of B2 to inhibit NF-kappaB could be valuable in a multi-drug approach.