NF-κB Reporter Gene Assay Research Articles

Regorafenib induces extrinsic and intrinsic apoptosis through inhibition of ERK/NF-κB activation in hepatocellular carcinoma cells.

The aim of the present study was to investigate the role of NF-κB inactivation in regorafenib-induced apoptosis in human hepatocellular carcinoma SK-HEP-1 cells. SK-HEP-1 cells were treated with different concentrations of the NF-κB inhibitor 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine (QNZ) or regorafenib for different periods. The effects of QNZ and regorafenib on cell viability, expression of NF-κB-modulated anti-apoptotic proteins and apoptotic pathways were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, DNA gel electrophoresis, flow cytometry and NF-κB reporter gene assay. Inhibitors of various kinases including AKT, c-Jun N-terminal kinase (JNK), P38 and extracellular signal-regulated kinase (ERK) were used to evaluate the mechanism of regorafenib-induced NF-κB inactivation. The results demonstrated that both QNZ and regorafenib significantly inhibited the expression of anti-apoptotic proteins and triggered extrinsic and intrinsic apoptosis. We also demonstrated that regorafenib inhibited NF-κB activation through ERK dephosphorylation. Taken all together, our findings indicate that regorafenib triggers extrinsic and intrinsic apoptosis through suppression of ERK/NF-κB activation in SK-HEP-1 cells.

Comparative evaluation of different cultivars of Flos Chrysanthemi by an anti-inflammatory-based NF-κB reporter gene assay coupled to UPLC-Q/TOF MS with PCA and ANN

Ethnopharmacological relevanceFlos Chrysanthemi (FC), a commonly used traditional Chinese medicine, has five major cultivars (“Boju”, “Chuju”, “Gongju”, “Hangbaiju” and “Huaiju”) from different sources. However, the active constituents of these cultivars have not been studied or characterized with respect to their bioactivity, which is a serious problem when considering quality and safety. Aim of the studyTo evaluate the differences among the five cultivars of FC, and to establish a method for the standardization and quality control of FC related to its bioactivity. Materials and methodsIn this study, the different ingredients in five cultivars of FC were identified by UPLC-Q/TOF and PCA, and the anti-inflammatory ingredients of FC were predicted and screened by artificial neural network (ANN) and an NF-κB luciferase reporter gene assay system. Using this comprehensive method, we successfully screened the anti-inflammatory markers of different cultivars of FC. ResultsNineteen marker ingredients were confirmed to contribute strongly to the cluster, and eleven compounds in the five cultivars of FC were found to exert potential anti-inflammatory effects. Among these compounds, the NF-κB inhibitor activity of apigenin-7-O-6″-malonyl-glucoside, luteolin-7-O-rutinoside, quercetin-7-O-galactoside, quercetin-3-O-glucoside, apigenin-7-O-rutinoside and apigenin-7-O-glucoside were first reported here. Chlorogenic acid, luteolin-7-O-glucoside, 3,5-dicaffeoylquinic acid and luteolin were confirmed to be the most important anti-inflammatory marker ingredients useful for the quality control of FC. ConclusionsThe proposed efficient and systematic method is helpful for the standardization and quality control of FC. Moreover, this comprehensive strategy may prove to be a powerful technique for the rapid establishment of quality control procedures related to bioactivity for other herbal samples and foods.

Pomegranate inhibits neuroinflammation and amyloidogenesis in IL-1β-stimulated SK-N-SH cells.

Pomegranate fruit, Punica granatum L. (Punicaceae), and its constituents have been shown to inhibit inflammation. In this study, we aimed to assess the effects of freeze-dried pomegranate (PWE) on PGE2 production in IL-1β-stimulated SK-N-SH cells. An enzyme immunoassay (EIA) was used to measure prostaglandin E2 (PGE2) production from supernatants of IL-1β-stimulated SK-N-SH cells. Expression of COX-2, phospho-IκB, and phospho-IKK proteins was evaluated, while NF-κB reporter gene assay was carried out in TNFα-stimulated HEK293 cells to determine the effect of PWE on NF-κB transactivation. Levels of BACE-1 and Aβ in SK-N-SH cells stimulated with IL-1β were measured with an in cell ELISA. PWE (25-200μg/ml) dose dependently reduced COX-2-dependent PGE2 production in SK-N-SH cells stimulated with IL-1β. Phosphorylation of IκB and IKK was significantly (p<0.001) inhibited by PWE (50-200μg/ml). Our studies also show that PWE (50-200μg/ml) significantly (p<0.01) inhibited NF-κB transactivation in TNFα-stimulated HEK293 cells. Furthermore, PWE inhibited BACE-1 and Aβ expression in SK-N-SH cells treated with IL-1β. Taken together, our study demonstrates that pomegranate inhibits inflammation, as well as amyloidogenesis in IL-1β-stimulated SK-N-SH cells. We propose that pomegranate is a potential nutritional strategy in slowing the progression of neurodegenerative disorders such as Alzheimer's disease.

How the venom from the ectoparasitoid Wasp nasonia vitripennis exhibits anti-inflammatory properties on mammalian cell lines.

With more than 150,000 species, parasitoids are a large group of hymenopteran insects that inject venom into and then lay their eggs in or on other insects, eventually killing the hosts. Their venoms have evolved into different mechanisms for manipulating host immunity, physiology and behavior in such a way that enhance development of the parasitoid young. The venom from the ectoparasitoid Nasonia vitripennis inhibits the immune system in its host organism in order to protect their offspring from elimination. Since the major innate immune pathways in insects, the Toll and Imd pathways, are homologous to the NF-κB pathway in mammals, we were interested in whether a similar immune suppression seen in insects could be elicited in a mammalian cell system. A well characterized NF-κB reporter gene assay in fibrosarcoma cells showed a dose-dependent inhibition of NF-κB signaling caused by the venom. In line with this NF-κB inhibitory action, N. vitripennis venom dampened the expression of IL-6, a prototypical proinflammatory cytokine, from LPS-treated macrophages. The venom also inhibited the expression of two NF-κB target genes, IκBα and A20, that act in a negative feedback loop to prevent excessive NF-κB activity. Surprisingly, we did not detect any effect of the venom on the early events in the canonical NF-κB activation pathway, leading to NF-κB nuclear translocation, which was unaltered in venom-treated cells. The MAP kinases ERK, p38 and JNK are other crucial regulators of immune responses. We observed that venom treatment did not affect p38 and ERK activation, but induced a prolonged JNK activation. In summary, our data indicate that venom from N. vitripennis inhibits NF-κB signaling in mammalian cells. We identify venom-induced up regulation of the glucocorticoid receptor-regulated GILZ as a most likely molecular mediator for this inhibition.

Glycyrrhetinic Acid Suppressed NF-κB Activation in TNF-α-Induced Hepatocytes

Tumor necrosis factor-alpha (TNF-α) is a crucial inflammatory cytokine when hepatocytes are damaged. Glycyrrhiza uralensis Fisch. (Chinese licorice) has been widely used in Chinese herbal prescriptions for the treatment of liver diseases and as a food additive. Nuclear factor-kappa B (NF-κB) reporter gene assay in TNF-α-induced HepG2 was used as a screening platform. IκBα phosphorylation and p65 translocation were measured by Western blotting, and nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression were further confirmed in rat primary hepatocytes. Results showed that TNF-α enhanced NF-κB activity was significantly attenuated by glycyrrhetinic acid in a concentration-dependent manner in the NF-κB reporter gene assay. Glycyrrhetinic acid decreased the gene expression of iNOS through inhibited IκBα phosphorylation and p65 translocation in protein level. Furthermore, NO production and iNOS expression were reduced by glycyrrhetinic acid in TNF-α-induced rat primary hepatocytes. These results suggest that glycyrrhetinic acid may provide hepatoprotection against chronic liver inflammation through attenuating NF-κB activation to alleviate the inflammation.

Extracts of the seaweed Sargassum macrocarpum inhibit the CpG-induced inflammatory response by attenuating the NF-κB pathway

The anti-inflammatory effects of extracts of the seaweed Sargassum macrocarpum (SME) in bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) were investigated. Primary BMDMs and BMDCs were used for cytokine production and western blot analysis. SME (0–50 μg/mL) pre-treatment resulted in a strong inhibitory effect against interleukin (IL)-12 p40, IL-6, and tumor necrosis factor (TNF)-α production in CpG-stimulated BMDMs and BMDCs. SME pre-treatment caused a strong inhibitory effect against NF-κB activation, as evaluated using degradation of IκBα and the NF-κB reporter gene assay. SME has significant anti-inflammatory properties and warrants further study regarding potential use as a medicinal food.

Functional assessment of the mutational effects of human IRAK4 and MyD88 genes

Human interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency and myeloid differentiating factor 88 (MyD88) deficiency syndromes are two primary immune-deficiency disorders with innate immune defects. Although new genetic variations of IRAK4 and MyD88 have recently been deposited in the single nucleotide polymorphism (SNP) database, the clinical significance of these variants has not yet been established. Therefore, it is important to establish methods for assessing the association of each gene variation with human diseases. Because cell-based assays, western blotting and an NF-κB reporter gene assay, showed no difference in protein expression and NF-κB activity between R12C and wild-type IRAK4, we examined protein–protein interactions of purified recombinant IRAK4 and MyD88 proteins by analytical gel filtration and NMR titration. We found that the variant of IRAK4, R12C, as well as R20W, located in the death domain of IRAK4 and regarded as a SNP, caused a loss of interaction with MyD88. Our studies suggest that not only the loss of protein expression but also the defect of Myddosome formation could cause IRAK4 and MyD88 deficiency syndromes. Moreover a combination of in vitro functional assays is effective for confirming the pathogenicity of mutants found in IRAK4 and MyD88-deficiency patients.

Identification and Comparison of Anti-Inflammatory Ingredients from Different Organs of Lotus Nelumbo by UPLC/Q-TOF and PCA Coupled with a NF-κB Reporter Gene Assay

Lotus nelumbo (LN) (Nelumbo nucifera Gaertn.) is an aquatic crop that is widely distributed throughout Asia and India, and various parts of this plant are edible and medicinal. It is noteworthy that different organs of this plant are used in traditional herbal medicine or folk recipes to cure different diseases and to relieve their corresponding symptoms. The compounds that are contained in each organ, which are named based on their chemical compositions, have led to their respective usages. In this work, a strategy was used to identify the difference ingredients and screen for Nuclear-factor-kappaB (NF-κB) inhibitors with anti-inflammatory ability in LN. Seventeen main difference ingredients were compared and identified from 64 samples of 4 different organs by ultra-performance liquid chromatography that was coupled with quadrupole/time of flight mass spectrometry (UPLC/Q-TOF-MS) with principal component analysis (PCA). A luciferase reporter assay system combined with the UPLC/Q-TOF-MS information was applied to screen biologically active substances. Ten NF-κB inhibitors from Lotus plumule (LP) extracts, most of which were isoquinoline alkaloids or flavone C-glycosides, were screened. Heat map results showed that eight of these compounds were abundant in the LP. In conclusion, the LP extracts were considered to have the best anti-inflammatory ability of the four LN organs, and the chemical material basis (CMB) of this biological activity was successfully validated by multivariate statistical analysis and biological research methods.

Sea lettuce (Ulva fasciata) extract has an inhibitory effect on proinflammatory cytokine production in CpG-stimulated bone marrow-derived macrophages and dendritic cells

This report was designed to study inhibitory effect of sea lettuce (Ulva fasciata) extract (UFE) on proinflammatory cytokine production in bone marrow-derived macrophages (BMDM) and dendritic cells (BMDC). The UFE (0–50 μg/mL) pre-treatment showed a dose dependant inhibitory effect on interlukin (IL)-12 p40, IL-6, and tumor necrosis factor (TNF)-α productions in CpG-stimulated BMDMs and BMDCs as compared to non-treated controls. The UFE pre-treatment exhibited strong inhibitory effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK) while it showed moderate inhibition on nuclear factor (NF)-κB activation as indirectly evaluated by degradation of IκBα. In activator protein (AP)-1 and NF-κB reporter gene assay, the UFE pre-treatment showed moderate inhibitory effect on both AP-1- and NF-κBdependent reporter gene activities. Thus, these results suggest that inhibitory effect of UFE on pro-inflammatory cytokine production may correlate with partial inhibition of both AP-1 and NF-κB pathways. Hence, our data warrant further studies concerning potentials of sea lettuce for medicinal food.

Inhibition of cell death of bone marrow-derived macrophages infected with Ehrlichia muris

Ehrlichia muris is a Gram-negative obligate intracellular bacterium belonging to the family Anaplasmataceae. It preferentially replicates inside macrophages by utilizing nutrients and processes of the host cell. In the present article, we studied the effects of E. muris infection on cell death of bone marrow-derived macrophages (BMDMs). Primary BMDMs were used for accessing E. muris-induced cell death, pro-inflammatory cytokine production and Western blot analysis. Human embryonic kidney cell line 293T (HEK293T) was used to access nuclear factor-kappaB (NF-κB) activity. BMDMs infected with E. muris showed significant inhibition of cell death when compared to uninfected cells. E. muris infection resulted in IκBα degradation, thus activation of NF-κB. In NF-κB reporter gene assay, the HEK293T cells infected with E. muris exhibited robust NF-κB-dependent luciferase activity in a bacterial dose-dependent manner. Furthermore, E. muris-induced inhibition of BMDMs cell death was abolished in the presence of MG132, a proteasome inhibitor that blocks NF-κB activation. Taken together, the results suggest that E. muris infection of BMDMs may have an inhibitory effect on cell death via a mechanism dependent on NF-κB activation.

Glucosamine Modulates TNF-α–Induced ICAM-1 Expression and Function Through O-Linked and N-Linked Glycosylation in Human Retinal Pigment Epithelial Cells

The purpose of this article was to investigate the effects of glucosamine (GlcN) on the TNF-α-induced expression of intercellular adhesion molecule 1 (ICAM-1) and the function of ICAM-1 in ARPE-19 cells in vitro. We quantified protein levels of TNF-α-induced ICAM-1 in ARPE-19 cells with Western blotting. The effects of GlcN on O-linked glycosylation, and therefore on ICAM-1 expression, were compared after the addition of alloxan, an inhibitor of O-linked N-acetylglucosamine transferase (OGT), or O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase (O-GlcNAcase [OGA]), or after OGT gene overexpression. The effect of GlcN on the N-linked glycosylation of ICAM-1 was evaluated by the change in its molecular mass on Western blotting. The effect of O-linked glycosylation on the nuclear factor κB (NF-κB) signaling pathway was examined using an NF-κB reporter gene assay. The effect of GlcN on ICAM-1 adhesion activity was examined using an ICAM-1 adhesion assay. GlcN, PUGNAc, and OGT overexpression inhibited TNF-α-induced ICAM-1 expression and NF-κB activity in ARPE-19 cells. Alloxan increased ICAM-1 expression and NF-κB activity in TNF-α-induced ARPE-19 cells. GlcN and tunicamycin reduced the molecular mass of TNF-α-induced ICAM-1 in ARPE-19 cells. The proteasome inhibitor MG-132 suppressed the GlcN-induced reduction in the molecular mass of TNF-α-induced ICAM-1. GlcN also attenuated the adhesion activity of TNF-α-induced ICAM-1. GlcN inhibits ICAM-1 expression and functions by modulating the O-linked glycosylation of factors involved in NF-κB signaling and by reducing the N-linked glycosylation of TNF-α-induced ICAM-1 in ARPE-19 cells. These effects may contribute to the GlcN-mediated anti-inflammatory effects in the eye.

Inhibitory effects of Carpinus tschonoskii leaves extract on CpG-stimulated pro-inflammatory cytokine production in murine bone marrow-derived macrophages and dendritic cells

This report describes the anti-inflammatory effects of MeOH extract from leaves of Carpinus tschonoskii (CE) on primary bone marrow-derived macrophage (BMDMs) and dendritic cells (BMDCs). Primary BMDMs and BMDCs were used for pro-inflammatory cytokine production and Western blot analysis. Human embryonic kidney cell line 293T (HEK293T) was used to access NF-κB activity. In all cases, CpG DNA was used to stimulate the cells. The CE (0-150μg/ml) was treated to BMDMs, BMDCs, and HEK293T cells. CE pre-treatment in CpG-stimulated BMDMs and BMDCs showed a dose-dependent inhibitory effect on pro-inflammatory cytokine (e.g., IL-12 p40, IL-6, and TNF-α) production as compared to non-treated controls. The CE pre-treatment had no significant inhibition on mitogen-activated protein kinases (MAPKs) phosphorylation but strongly inhibited IκBα degradation. In NF-κB reporter gene assay, the CE pre-treatment inhibited NF-κB-dependent luciferase activity in a dose-dependent manner. Taken together, these data suggest that CE has significant inhibitory effect on pro-inflammatory cytokine production and warrant further studies concerning potentials of CE for medicinal uses.

Anti-inflammatory Activity ofCarpinus tschonoskiiLeaves Extract in R848-stimulated Bone Marrow-derived Macrophages and Dendritic Cells

The present study aims to evaluate the anti-inflammatory effect of methanol extract from leaves of Carpinus tschonoskii (CE) on R848-stimulated primary bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs). Primary BMDMs and BMDCs were used for pro-inflammatory cytokine production. Human embryonic kidney cell line 293T (HEK293T) was used to access NF-κB activity. In all cases, R848 was used to stimulate the cells. The CE (0~150 ㎍/㎖) was treated to BMDMs, BMDCs, and HEK293T cells. CE pre-treatment in R848-stimulated BMDMs and BMDCs showed a dose-dependent inhibitory effect on pro-inflammatory cytokine (e.g., IL-12 p40, IL-6, and TNF-α) production as compared to non-treated controls. In NF-κB reporter gene assay, the CE pre-treatment inhibited NF-κBdependent luciferase activity in a dose-dependent manner. Overall, our findings suggest that CE has significant inhibitory effect on pro-inflammatory cytokine production and deserve further studies concerning potentials of CE for medicinal uses.

Suppression of human prostate cancer PC-3 cell growth by N-acetylcysteine involves over-expression of Cyr61

N-acetylcysteine (NAC), sulfidryl-containing thiol antioxidant, has been heralded as chemopreventive agent, generally because of its ability to scavenge free radicals. It also suppresses the proliferation of many cancer cells; however, the antiproliferative mechanism(s) remain to be fully elucidated. In this study, we investigated a growth-suppressive mechanism of NAC action in androgen-independent prostate carcinoma PC-3 cells. NAC (⩾1 mM) inhibited the proliferation of PC-3 cells in a dose- and time-dependent manner. Moreover, NAC treatment suppressed the activation of NF-κB induced by IKK-β as detected by the NF-κB reporter gene assay. NAC exerted a biphasic effect on the intracellular ROS levels depending on incubation time; the antioxidant effect was seen within 2 h after NAC treatment, however, a pro-oxidant effect was evident after 48 h treatment. In addition to these effects, NAC treatment elicited a dose- and time-dependent increase in the Cyr61 expression that was accompanied by an increase in its mRNA and blocked by cycloheximide pretreatment. Importantly, NAC treatment caused an early but transient activation of Akt and Erk1/2. The NAC-induced increase in Cyr61 protein levels was suppressed by the PI3K inhibitor (Ly294002) and, to a lesser extent, MEK/Erk1/2 inhibitor (PD98059). Taken together, our data suggest that the antiproliferative effect of NAC is partially mediated by intracellular ROS production, the inhibition of NF-κB activity, and the activation of PI3K- and/or MEK/Erk-related intracellular signaling pathways, which lead to up-regulation of Cyr61 expression.

The discovery of thienopyridine analogues as potent IκB kinase β inhibitors. Part II

An SAR study that identified a series of thienopyridine-based potent IκB Kinase β (IKKβ) inhibitors is described. With focuses on the structural optimization at C 4 and C 6 of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C 4, whereas polar groups with proper orientation at C 6 efficiently enhance compound potency. The most potent analogues inhibit IKKβ with IC 50s as low as 40 nM, suppress LPS-induced TNF-α production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-κB reporter gene assay, demonstrating that they directly interfere with the NF-κB signaling pathway.

Anti-inflammatory Effects of the Methanol Extract of Polytrichum Commune via NF-κB Inactivation in RAW 264.7 Macrophage Cells

Abstarct − As an attempt to search for bioactive natural products exerting anti-inflammatory activity, we evaluated the effects of the methanol extract of Polytrichum commune Hedw (PCM) (Polytrichaceae) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E 2 (PGE 2 ) and pro-inflammatory cytokines release in murine macrophage cell line RAW 264.7. PCM potently inhibits the production of NO, PGE 2 , tumor necrosis factor (TNF)-α and interleukin (IL)-6. Consistent with these results, PCM also concentration-dependently inhibited LPS-induced inducible NO synthase (iNOS) and cyclooxygase (COX)-2 at the protein levels, and iNOS, COX-2, TNF-α and IL-6 at the mRNA levels without an appreciable cytotoxic effect on RAW 264.7 macrophage cells. Furthermore, PCM inhibited LPS-induced nuclear factor-kappa B (NF-κB) activation as determined by NF-κB reporter gene assay, and this inhibition was associated with a decrease in the nuclear translocation of p65 and p50 NF-κB. Taken together, these results suggest that PCM may play an anti-inflammatory role in LPS-stimulated RAW 264.7 macrophages through the inhibitory regulation of iNOS, COX-2, TNF-α and IL-6 via NF-κB inactivation.

The novel non-boronic proteasome inhibitor S-2209 induces apoptosis and growth arrest in multiple myeloma cells

8581 Background: Bortezomib is the first approved member of a new class of antineoplastic agents, the proteasome inhibitors. Anti- myeloma activity of Bortezomib monotherapy is only moderate and specific toxicity often limits its clinical use. Further proteasome inhibitors need to be developed. Methods: S-2209 was characterized by several assays. Inhibition of the chymotryptic activity of the human 20S proteasome was determined with the in-vivo protease inhibition assay. Additionally, proteasome inhibition was determined in isolated PBMCs from S2209-pretreated wistar rats. Inhibition of NFκB activity was determined using a NFκB reporter gene assay. Cell growth rates of MM cells were measured with the WST-1 assay. Induction of apoptosis was shown by annexin-V-FITC staining. Intracellular signal modulation was demonstrated by western blotting. Toxicity of the substance was tested in male wistar rats. Results: The proteasome inhibition assay revealed an IC50 at ∼220nM. The NFκB inhibition assay using an A549-NFκB-SEAP transfected cell line showed an EC50 of 0.9μM. Upon incubation with S-2209, cell growth as well as cell proliferation in MM cell lines was significantly inhibited (IC50 100nM - 600nM). Furthermore, the incubation with S-2209 resulted in strong induction of apoptosis in all four MM cell lines (IC50 at ∼300nm) as well as primary cells. Western blotting revealed caspase-3 cleavage and upregulation of p53 and increased phosphorylation of IκB. No induction of apoptosis was detected in PBMCs from healthy humans. Despite the administration of 5, 10 or 15mg/kg/day in wistar rats, no toxicity with respect to body weight, hepatic enzymes (ALAT ASAT, ALP), creatinin or hemoglobin was seen. Proteasome inhibition in white blood cells isolated from the treated rats was higher in the S-2209 treated animals than in control animals treated with 0.1mg/kg/d bortezomib (89% vs. 70% respectively). Conclusions: The proteasome inhibitor S-2209 inhibitis MM cell growth and induces apoptosis. This is accompanied by a strong inhibition of proteasome and of the NFκB activity. Because S-2209 shows a favourable toxicity profile in vivo, further clinical development of this promising drug is urgently needed. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration 4SC

The type III secretion system-dependent repression of NF-κB activation to the intracellular growth of Edwardsiella tarda in human epithelial cells

Edwardsiella tarda is a pathogen with a broad host range infecting animals and humans. We have reported recently that the type III secretion system (TTSS) is essential for intracellular replication of the bacterium in murine macrophages. The present study shows that the TTSS is also needed for intracellular growth of the bacterium in human epithelial cells (HEp-2). However, different from the previous microarray analyses on murine macrophages, upregulation of the mRNA expression level of NF-kappaB target genes was not detected in the infected HEp-2 cells. The wild-type E. tarda, but not its TTSS mutant, actually repressed the tumor necrosis factor alpha-dependent NF-kappaB activation in an NF-kappaB reporter gene assay. These results suggest TTSS-dependent repression of the NF-kappaB activation in HEp-2 cells infected with E. tarda.

Proteasome inhibition by peptide-semicarbazones

Peptide-semicarbazones derived from Z-Trp-Trp-Phe-aldehyde inhibit the chymotryptic activity of the human proteasome at nanomolar concentrations, but are less active in a NFκB reporter gene assay. Cyclic semicarbazones, in contrast, combine a strong inhibitory effect on the enzyme with an inhibition of NFκB signaling in the nanomolar range. In addition, a practical synthesis for scale-up of such compounds was developed.

Novel heterocycle-substituted pyrimidines as inhibitors of NF-κB transcription regulation related to TNF-α cytokine release

Novel heterocyclic ring-substituted pyrimidines have been designed as inhibitors of glycogen synthase kinase-3β (GSK-3β) from the modification of known inhibitors. Several potent inhibitors exhibiting nanomolar activities were discovered against GSK-3β kinase as well as in an NF-κB reporter gene assay. Based on the results from in vitro TNF-α release inhibition and in vivo endotoxima, these inhibitors are expected to be useful candidates for treatment of inflammation-related diseases.