Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

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.     

Effect of thymopentin on the production of cytokines, heat shock proteins, and NF-κB signaling proteins

In vivo effects of thymopentin, an active fragment of the naturally occurring thymic hormone thymopoietin, on the production of cytokines, nitric oxide, heat shock proteins, and signaling proteins NF-κB, phNF-κB, and IκB-α in lymphoid cells of male NMRI mice was studied. Activation of production of several cytokines (IL-1α, IL-2, IL-6, IL-10, and IFN-γ), nitric oxide, and heat shock proteins (HSP70 and HSP90) was observed in peritoneal macrophages and spleen lymphocytes of mice that received intraperitoneal injections of thymopentin (15μg per 100 g body weight). Thymopentin apparently produces stress-like rather than damaging effects. A probable action mechanism of this hormone is activation of the NF-κB signaling pathway, which is most pronounced at the NF-κB phosphorylation stage.     

Inhibition of IκB kinase-β and IκB kinase-α by heterocyclic adamantyl arotinoids

We recently reported on a series of retinoid-related molecules containing an adamantyl group, a.k.a. adamantyl arotinoids (AdArs), that showed significant cancer cell growth inhibitory activity and activated RXRα (NR2B1) in transient transfection assays while devoid of RAR transactivation capacity. We have now explored whether these AdArs could also bind and inhibit IKKβ, a known target that mediates the induction of apoptosis and cancer cell growth inhibition by related AdArs containing a chalcone functional group. In addition, we have prepared and evaluated novel AdArs that incorporate a central heterocyclic ring connecting the adamantyl-phenol and the carboxylic acid at the polar termini. Our results indicate that the majority of the RXRα activating compounds lacked IKKβ inhibitory activity. In contrast, the novel heterocyclic AdArs containing a thiazole or pyrazine ring linked to a benzoic acid motif were potent inhibitors of both IKKα and IKKβ, which in most cases paralleled significant growth inhibitory and apoptosis inducing activities.     

Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.     

Molecular activation of NF-κB, pro-inflammatory mediators, and signal pathways in γ-irradiated mice

The effects of γ-irradiation on inflammatory gene expression, including NF-κB activation, in the kidney of C57/BL6 mice exposed to 1–9 Gy doses of ⁶⁰Co γ-irradiation. Radiation enhanced the NF-κB activation and oxidative stress caused a dose-dependent disruption in the redox balance. The significance of this study is the new molecular information gained on γ-irradiation effects through the activation of pro-inflammatory genes by NF-κB via the MAPK signaling pathway. Considering the exquisite sensitivity of NF-κB and other pro-inflammatory mediators to the redox status, we conclude that the activation of inflammatory processes by irradiation is likely initiated by increased oxidative stress.