Novel Inhibitors of Cytokine-induced IκBα Phosphorylation and Endothelial Cell Adhesion Molecule Expression Show Anti-inflammatory Effects in Vivo

Abstract

We have identified two compounds that inhibit the expression of endothelial-leukocyte adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor-␣-induced phosphorylation of IB-␣, resulting in decreased nuclear factor-B and decreased expression of adhesion molecules. The effects on both IB-␣ phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC 50 of approximately 10 M. These agents selectively and irreversibly inhibited the tumor necrosis factor-␣-inducible phosphorylation of IB-␣ without affecting the constitutive IB-␣ phosphorylation. Although these compounds exhibited other activities, including stimulation of the stress-activated protein kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a 130 -140-kDa protein, these effects are probably distinct from the effects on adhesion molecule expression since they were reversible. One compound was evaluated in vivo and shown to be a potent anti-inflammatory drug in two animal models of inflammation. The compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a rat adjuvant arthritis model. These studies suggest that inhibitors of cytokine-inducible IB␣ phosphorylation exert anti-inflammatory activity in vivo.The adhesion of circulating leukocytes to vascular endothelium is critical to inflammatory responses (reviewed in Refs. 1-3). Interaction of the selectin family of adhesion proteins and lectin counter-receptors is the predominant mechanism mediating initial adhesion between leukocytes and the vessel wall. The expression of endothelial-leukocyte adhesion molecule-1 (E-selectin, CD62E), vascular cell adhesion molecule-1 (VCAM-1, 1 CD106), and intercellular adhesion molecule-1 (ICAM-1, CD54) on the surface of endothelial cells is elevated at sites of inflammation (2, 4). Induction of these molecules by tumor necrosis factor-␣ (TNF␣) and other inflammatory cytokines is regulated at the level of gene transcription and requires binding of the transcription factor nuclear factor-B (NF-B) to the regulatory regions within the promoters of each of these genes (5-12).The NF-B/Rel transcription factor family plays an important role in cytokine-induced gene activation (13-15). The Rel family includes p50 (NFKB1), p52 (NFKB2), p65 (RelA), RelB, v-Rel, and c-Rel. In endothelial cells, the p50⅐p65 heterodimer is the predominant species that binds to B consensus sequences in the VCAM-1, ICAM-1, and E-selectin genes and activates gene transcription. NF-B is located in the cytoplasm of cells in an inactive form in association with the inhibitor IB-␣. In response to TNF␣ stimulation, IB-␣ is phosphorylated on 2 serine residues (Ser-32 and Ser-36), ubiquitinated, and degraded by a proteosome-dependent pathway allowing active NF-B to translocate to the nucleus where it can activate gene expression (16 -23). Many NF-B-dependent genes including the adhesion m...

Antitumor activity and macrophage nitric oxide producing action of medicinal herb, Crassocephalum crepidioides

“…Because activation of the iNOS promoter by C. crepidioides required the activation of NF-κB, we blocked NF-κB activation with Bay 11-7082, an inhibitor of IκBα phosphorylation [24] or LLnL, a proteasome inhibitor [25]. Bay 11-7082 and LLnL reduced C. crepidioides -induced iNOS promoter activity (Figure 8A).…”

“…Bay 11-7082 and LLnL are relatively specific inhibitors of NF-κB activation [24,25]. Both agents blocked the promoter activity of iNOS, the expression of iNOS mRNA, and the production of nitrite, indicating the likely involvement of NF-κB in the induction of not just iNOS-driven reporter constructs but also the iNOS gene itself in C. crepidioides -treated macrophages.…”

NF‐κB inhibition reverses acidic bile‐induced miR‐21, miR‐155, miR‐192, miR‐34a, miR‐375 and miR‐451a deregulations in human hypopharyngeal cells

“…Specifically, the combination of bile and acid (pH ≤4.0) constitutively activates NF‐κB, up‐regulating the expression of cancer‐related genes and deregulating the expression of oncogenic miRNA markers, such as “oncomirs” miR‐21, miR‐192, miR‐155 and “tumour suppressors” miR‐34a, miR‐375 and miR‐451a, in pre‐malignant lesions of murine laryngopharyngeal mucosa 9, 10. Here, we describe an in vitro model exploring repetitive exposures of normal human hypopharyngeal cells to acidic bile, with and without BAY 11‐7082, a pharmacologic inhibitor of NF‐κB 11. We hypothesize that NF‐κB inhibitor is capable of preventing the acidic bile‐induced up‐regulation of “oncomirs” miR‐21, miR‐155 and miR‐192 and down‐regulation of “tumour suppressor” miR‐34a, miR‐375 and miR‐451a, previously associated with laryngopharyngeal cancer,12, 13, 14, 15, 16, 17 providing insight into interactions of transcriptionally active NF‐κB with cancer‐related miRNA markers.…”

“…BAY 11‐7082 was selected as a reliable inhibitor of NF‐κB pathway that has been widely used in many studies exploring the effect of NF‐κB 11, 24, 25. It has been suggested that BAY 11‐7082 offers the most rapid and potent antitumour effect among other NF‐κB inhibitors 24 and can possibly be used as a sensitizer of anticancer therapy,26, 27 increasing the intrinsic susceptibility of cancer cells to chemotherapeutic agents 28…”

The L1 major capsid protein of HPV16 differentially modulates APC trafficking according to the vaccination or natural infection context

“…1B). DC pretreated with BAY11-7082, an inhibitor of IkBa processing [18], and incubated with VLP or LPS also showed a decreased nuclear expression of p65 compared with control DC. By flow cytometry and by Western blotting, BAY11-7082 was also found to have an inhibitory effect on CXCR4 expression in DC stimulated by VLP or LPS compared with DC activated by VLP or LPS alone ( Fig.…”