Adriamycin induces myocardium apoptosis through activation of nuclear factor κB in rat

Adriamycin is one of the most effective and useful antineoplastic agents. Acute doxorubicin cardiotoxicity involved cardiomyocyte apoptosis. In this study, we investigated whether adriamycin induced myocardium apoptosis through activation of nuclear factor κB in rat. Forty male Wistar rats were randomly divided into five groups: control, ADR 5 mg/kg, ADR 10 mg/kg, ADR 15 mg/kg group and ADR + PDTC 200 mg/ml group. Myocardial apoptosis was detected by DNA fragmentation assay and TUNEL assay; Location and distribution of p-IκBα was observed by immunohistochemical assay; Myocardial expression of p-IκBα protein was assessed by Western blot analysis; Activity of NF-κB was evaluated by Electrophoretic Mobility Shift Assay. The myocardial apoptotic index, expression of p-IκBα, and binding activity of NF-κB increased significantly in ADR groups in dose-dependent manner. PDTC as a nonspecific inhibitor of NF-κB protected myocardium from apoptosis by inhibiting NF-κB activation. Adriamycin induces myocardium apoptosis through activation of nuclear factor κB in rat and NF-κB activation requires IκBα degradation.     

Nitric Oxide Potentiates TNF-α-Induced Neurotoxicity Through Suppression of NF-κB

Modulation of enzyme activity through nitrosylation has recently been identified as a new physiological activity of nitric oxide (NO). We hypothesized that NO enhances the TNF-α-induced death of retinal neurons through a suppression of nuclear factor-κB (NF-κB) by nitrosylation. In this study, cells from the RGC-5 line were exposed to different concentrations (2.0, 10, and 50 ng/ml) of TNF-α, and the degree of TNF-α-induced cell death was determined by the WST-8 assay and by flow cytometric measurements of the externalization of phosphatidylserine. The effects of etanercept, a soluble TNFR-Fc fusion protein, and S-nitroso-N-penicillamine (SNAP), an NO donor, on the toxicity were determined. Experiments were also performed to determine whether nitric oxide synthase (NOS) was associated with the toxicity of TNF-α. The activation of NF-κB was determined by the detection of the p65 subunit in the nuclear extracts. Our results showed that exposure of RGC-5 cells to different concentrations of TNF-α significantly decreased the number of living cells in a dose-dependent way. The death was partially due to apoptosis with an externalization of phosphatidylserine, and the death was suppressed by etanercept. Exposure to TNF-α increased the activation of NF-κB and the expression of iNOS. Although NF-κB inhibitors suppressed the increase of iNOS, they also potentiated the TNF-α-induced death. Both L-NAME and aminoguanidine, both NOS inhibitors, rescued the cells from death. In contrast, addition of SNAP caused nitrosylation of the inhibitory κB kinase, and suppressed the NF-κB activation and potentiated the TNF-α-induced neurotoxicity. These results indicate that NO potentiates the neurotoxicity of TNF-α by suppressing NF-κB.     

Effect of a small molecule inhibitor of nuclear factor-κB nuclear translocation in a murine model of arthritis and cultured human synovial cells

A small cell-permeable compound, dehydroxymethylepoxyquinomicin (DHMEQ), does not inhibit phosphorylation and degradation of IκB (inhibitor of nuclear factor-κB [NF-κB]) but selectively inhibits nuclear translocation of activated NF-κB. This study aimed to demonstrate the antiarthritic effect of this novel inhibitor of the NF-κB pathway in vivo in a murine arthritis model and in vitro in human synovial cells. Collagen-induced arthritis was induced in mice, and after onset of arthritis the mice were treated with DHMEQ (5 mg/kg body weight per day). Using fibroblast-like synoviocyte (FLS) cell lines established from patients with rheumatoid arthritis (RA), NF-κB activity was examined by electrophoretic mobility shift assays. The expression of molecules involved in RA pathogenesis was determined by RT-PCR, ELISA, and flow cytometry. The proliferative activity of the cells was estimated with tritiated thymidine incorporation. After 14 days of treatment with DHMEQ, mice with collagen-induced arthritis exhibited decreased severity of arthritis, based on the degree of paw swelling, the number of swollen joints, and radiographic and histopathologic scores, compared with the control mice treated with vehicle alone. In RA FLS stimulated with tumor necrosis factor-α, activities of NF-κB components p65 and p50 were inhibited by DHMEQ, leading to suppressed expression of the key inflammatory cytokine IL-6, CC chemokine ligand-2 and -5, matrix metalloproteinase-3, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. The proliferative activity of the cells was also suppressed. This is the first demonstration of an inhibitor of NF-κB nuclear translocation exhibiting a therapeutic effect on established murine arthritis, and suppression of inflammatory mediators in FLS was thought to be among the mechanisms underlying such an effect.     

Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells

Background  

Activation of nuclear factor-κB (NF-κB) is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α) induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1) can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50).     

IKKγ (NEMO) is involved in the coordination of the AP-1 and NF-κB pathways

Tumor necrosis factor alpha (TNFα) activates the nuclear factor-kappaB (NF-κB) pathway in various cell types, leading to expression of cell survival and inflammatory proteins. One mechanism of cell survival brought about by NF-κB is the inhibition of Activator Protein-1 (AP-1), which when activated, could lead to cell death. However, TNFα can also induce the AP-1 pathway, and the mechanisms by which these two pathways are regulated in response to TNFα are poorly understood. We proposed that Inhibitor of κB Kinase gamma (IKKγ) (which is also known as NF-κB essential modulator, NEMO) plays a key role in integrating and coordinating these two pathways. Our results showed that IKKγ activates the AP-1 pathway, via a mechanism that is dependent on the first leucine zipper (LZ) domain of IKKγ, by interacting with two proteins of the AP-1 complex, c-Jun and c-Fos, and changing the phosphorylation status of c-Jun. Even though IKKγ is required for the activation of NF-κB, we found that it reduced the activity of NF-κB when it was overexpressed. In summary, we demonstrated that transfected IKKγ, while inhibiting the NF-κB pathway, directly interacts with the AP-1 proteins and activates the AP-1 pathway independent of its effects on NF-κB. Our results indicate that IKKγ regulates TNFα signaling by coordinating cell responses mediated by the AP-1 and NF-κB pathways. A. S. Shifera and J. M. Friedman contributed equally to this article. Marshall S. Horwitz—Deceased: This article is dedicated to his loving memory.     

Effects of glutamine on the nuclear factor-kappaB signaling pathway of murine peritoneal macrophages

The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappaB (NF-κB) signaling pathway of murine peritoneal macrophages. Since glutamine is essential for the normal functioning of macrophages, it was hypothesized that in vitro glutamine supplementation would increase NF-κB activation. Peritoneal macrophages were pretreated with glutamine (0, 0.6, 2 and 10 mM) before incubation with lipopolysaccharide (LPS), and the effects of glutamine on the production of tumor necrosis factor-alpha and on the expression and activity of proteins involved in the NF-κB signaling pathway were studied by an enzyme linked immuno-sorbent assay, Western blotting, and an electrophoretic mobility shift assay. Glutamine treatment (2 and 10 mM) increased the activation of NF-κB in LPS-stimulated peritoneal macrophages (P < 0.05). In non-stimulated cells, glutamine treatment (2 and 10 mM) significantly reduced IκB-α protein expression (P < 0.05). Glutamine modulates NF-κB signaling pathway by reducing the level of IκB-α, leading to an increase in NF-κB within the nucleus in peritoneal macrophages.     

Salsolinol,an Endogenous Neurotoxin,Activates JNK and NF-κB Signaling Pathways in Human Neuroblastoma Cells

Salsolinol, an endogenous neurotoxin, is known to be involved in the pathogenesis of Parkinson’s disease (PD). In the present study, we have investigated the effects of salsolinol on the activation of two different signaling pathways that involve c-Jun N-terminal kinase (JNK), and nuclear factor-κB, (NF-κB) in human dopaminergic neuroblastoma SH-SY5Y cells. Salsolinol treatment caused upregulation in the levels of c-Jun and phosphorylated c-Jun. It also caused degradation of IκBα and translocated the active NF-κB into the nucleus. The binding activity of NF-κB to DNA was enhanced by salsolinol in a concentration dependent manner. Furthermore, salsolinol decreased the levels of the anti-apoptotic protein Bcl-2, and increased pro-apoptotic protein Bax, while enhancing the release of cytochrome-c from mitochondria. Mitochondrial complex-I activity was significantly decreased and reactive oxygen species (ROS) were increased in salsolinol treated cells. These results partly suggest that salsolinol-induced JNK and NF-κB signaling pathways may be involved in induction of apoptosis in human dopaminergic neurons, as seen in Parkinson’s disease.