1,8-Cineol inhibits nuclear translocation of NF-κB p65 and NF-κB-dependent transcriptional activity

Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear.Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells.Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases.     

Thiol modulation of TNFα and IL-1 induced MnSOD gene expression and activation of NF-κB

TNF and IL-1 each can activate NF-B and induce gene expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix enzyme which can provide critical protection against hyperoxic lung injury. The regulation of MnSOD gene expression is not well understood. Since redox status can modulate NF-B and potential B site(s) exist in the MnSOD promoter, the effect of thiols (including NAC, DTT and 2-ME) on TNF and IL-1 induced activation of NF-B and MnSOD gene expression was investigated. Activation of NF-kB and increased MnSOD expression were potentiated by thiol reducing agents. In contrast, thiol oxidizing or alkylating agents inhibited both NF-B activation and elevated MnSOD expression in response to TNF or IL-1. Since protease inhibitors TPCK and TLCK can inhibit NF-B activation, we also investigated the effect of these compounds on MnSOD expression and NF-B activation. TPCK and TLCK each inhibited MnSOD gene expression and NF-B activation. Since the MnSOD promoter also contains anAP-1 binding site, the effect of thiols and thiol modifying agents on AP-1 activation was investigated. Thiols had no consistent effect onAP-1 activation. Likewise, some of the thiol modifying compounds inhibited AP-1 activation by TNF or IL-1, whereas others did not. Since diverse agents had similar effects on activation of NF-B and MnSOD gene expression, we have demonstrated that activation of NF-B and MnSOD gene expression are closely associated and that reduced sulfhydryl groups are required for cytokine mediation of both processes.Abbreviations O₂ Superoxide radical - H₂O₂ Hydrogen peroxide - NAC N-acetyl L-cysteine - DTT Dithiothreitol - 2-ME 2-Mercaptoethanol - MnSOD Manganese superoxide dismutase - NF-B Nuclear factor kappa B - AP-1 Activator protein-1 - NBT Nitroblue tetrazolium - CAT Chloramphenicol acetyltransferase - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - TLCK Na-p-tosyl-L-lysine chloromethyl ketone - TAME N--p-tosyl-L-arginine methyl ester - NEM N-ethyl maleimide - DEM Diethyl maleate - CDNB 1-chloro-2,4-dinitrobenzene - DTT_OX Oxidized dithiothreitol     

Acyl derivatives of boswellic acids as inhibitors of NF-κB and STATs

Boswellic acid acylates including their epimers were synthesized and screened against a panel of human cancer cell lines. They exhibited a range of cytotoxicity against various human cancer cell lines thereby leading to the development of a possible SAR. One of the identified lead compounds was found to be an inhibitor of the NF-κB and STAT proteins, warranting further investigations to be developed into a potential anticancer lead.     

Association of PARP-1, NF-κB,NF-κBIA and IL-6, IL-1β and TNF-α with Graves Disease and Graves Ophthalmopathy

Background

Graves Disease (GD) is an autoimmune disorder affected by an interaction of multiple genes such as Nuclear Factor-κB (NF-κB), Nuclear Factor-κB Inhibitor (NF-κBIA), Poly (ADP-ribose) polymerase-1 (PARP-1) and cytokines like Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) and mostly accompanied by an ocular disorder, Graves Ophthalmopathy (GO). We hypothesize that there is a relationship between GD, GO, polymorphisms of inflammatory related genes and their association with cytokines, which may play important roles in autoimmune and inflammatory processes.

Subjects and methods

To confirm our hypothesis, we studied the polymorphisms and cytokine levels of 120 patients with GD and GO using PCR-RFLP and ELISA methods, respectively.

Results

We found that patients with GG genotype and carriers of G allele of PARP-1 G1672A polymorphism are at risk in the group having GD (p = 0.0007) while having GA genotype may be protective against the disease. PARP-1 C410T polymorphism was found to be associated with GO by increasing the risk by 1.7 times (p = 0.004). Another risk factor for development of GO was the polymorphism of del/ins of NFkB1 gene (p = 0.032) that increases the risk by 39%. Levels of cytokines were also elevated in patients with GD, but no association was found between levels of cytokines and the development of GO as there was no change in levels of cytokines.

Conclusions

We suggest that, PARP-1 and NFkB1 gene polymorphisms may be risk factors for developing Graves Disease and Ophthalmopathy.     

Effects of receptor activator of NF-κB ligand gene silencing on the human osteoblast-like MG63 cells

The osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of NF-κB (RANK) system plays an important role in the pathogenesis of metabolic bone diseases. This study is aimed to investigate effects and mechanisms of RANKL gene silencing on the function of human osteoblast-like MG63 cells by RNA interference using a lentivirus-based small hairpin RNA (vshRNA) delivery system. After RANKL-specific vshRNAs were designed, constructed and transfected into MG63 cells, changes in the expression levels of RANKL mRNA and protein were determined by Western blot and RT-PCR, respectively; changes in cell activity and cell cycle distribution were examined by thiazolyl blue tetrazolium bromide assay and flow cytometry. The expression levels of RANKL mRNA and protein in MG63 cells were reduced by transferring RANKL-specific vshRNAs. Compared to cells infected with negative control virus, the proliferation of cells infected with the recombinant virus was more likely to be inhibited. Furthermore, the cell cycle of MG63 was altered, with the number of G1 phase cells decreasing significantly (P < 0.05). RANKL-specific vshRNAs can significantly inhibit the expression of the target gene in MG63 cells. RANKL gene silencing can inhibit the proliferation and alter the cell cycle of MG63 cells. Our findings suggest that RANKL might play an important role in the regulation of growth and cell cycle of MG63 cells.     

Theoretical studies on pyrimidine substituent derivatives as dual inhibitors of AP-1 and NF-κB

Theoretical studies on the three-dimensional (3D) quantitative structure-activity relationship (QSAR) and mechanisms of action of a series of pyrimidine substituent derivatives as dual inhibitors of AP-1 and NF-κB were carried out using comparative molecular field analysis (CoMFA) and docking methods. The established 3D-QSAR model exhibits a satisfying statistical quality and prediction ability. Docking results show somewhat lower average values of the flexible and rigid energy scores in the chosen binding sites. The docking analysis offers appropriate orientations and conformations of these compounds at the binding sites to both AP-1 and NF-κB in good agreement with the 3D-QSAR model from CoMFA. The combined CoMFA and docking study suggests the following substituent selections: substituent R₂ should be a kind of H–N–thienyl or CH₃–N–thienyl group; substituent R₅ should be a kind of COO–tBu or COOEt group; and substituent R₄ should be a CH₂CH₃ or 2-thienyl group. The docking analysis also shows that the binding sites fall just at the joint regions between AP-1 (or NF-κB) and DNA, where these compounds can effectively prevent free AP-1 and NF-κB from binding to DNA, and this may be the reason that derivatives with pyrimidine substituents have an inhibition function. In addition, a very interesting finding was that the binding sites of both AP-1 and NF-κB have a common structural characteristic, thereby providing a reasonable explanation for the dual inhibition functions of these compounds towards both AP-1 and NF-κB. These theoretical results help to deepen our understanding of the inhibition mechanism of these pyrimidine substituent derivatives, and will aid in directing further drug-molecular design.     

Fungal substances as modulators of NF-κB activation pathway

MCF7 breast cancer cell line, carrying a luciferase reporter gene under the control of nuclear factor kappa B (NF-κB)-responsive promoter, was established and used for the screening of fungal organic extracts for their ability to interfere with the NF-κB activation pathway. Twenty-eight crude fungal extracts, out of 242, were found to inhibit NF-κB reporter activity by more than 40%. Furthermore, positive extracts were used to evaluate their antiproliferative activity as well as their ability to influence the phosphorylation and degradation levels of IκBa. Fungal extracts prepared from Marasmius oreades and Cyathus striatus showed significant inhibitory effects on the NF-κB activation pathway. Taken together, our results support the notion of the presence of novel activities that might be utilized as cancer therapeutics.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

Rugulactone derivatives act as inhibitors of NF-κB activation and modulates the transcription of NF-κB dependent genes in MDA-MB-231cells

Rugulactone and its analogues were synthesized following Horners–Wadsworth–Emmons and ring-closing metathesis as the key reactions. A library of new rugulactone analogues were designed, synthesized and evaluated for their anticancer activity in breast cancer cells. All analogues have shown anti-proliferative activity, while some of them exhibited significant cytotoxicity. In assays related to cell-cycle distribution, these conjugates induced G1 cell-cycle arrest in MDA-MB-231 cells. The cell cycle arrest nature was further confirmed by examining the effect on Cyclin E and Cdk2 proteins that acts at G1-S phase transition. Immunocytochemistry assay revealed that these compounds inhibited nuclear translocation of NF-κB protein, thereby activation of NF-κB was inhibited. The expression of NF-κB target genes such as Cyclin D1 and Bcl-xL were severely affected. Apart from acting on NF-κB, these compounds also regulate class I Histone deacetylase proteins such as (HDAC-3 and 8) that have a crucial and regulatory role in cell-proliferation. Simultaneously, the apoptotic inducing nature of these compounds was confirmed by activation of PARP protein, a protein that plays a key role in DNA damage and repair pathways. Among all compounds of this series 3g is the most potent compound and can be used for further studies.