Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

Maikho Thoh,Pankaj Kumar,Hampathalu A Nagarajaram,Sunil K Manna

doi:10.1074/jbc.m109.065847

Abstract

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

Highlights

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on Tumor necrosis factor (TNF)-induced cell signaling in human cell lines was investigated
We examined the effect of azadirachtin on TNFinduced biological responses in different cell types
Azadirachtin Inhibits TNF-induced Cell Signaling—TNF, a potent inducer of cell signaling that leads to nuclear factor ␬B (NF-␬B) activation, can induce cell death either by inducing expression of proapoptotic genes and/or recruitment of death domain-containing proteins

Summary

EXPERIMENTAL PROCEDURES

Materials—Tissue culture chemicals were obtained from Invitrogen (Grand Island, NY). Azadirachtin and most of the general chemicals were obtained from Sigma Aldrich Chemicals. Azadirachtin Interacts with the TNF Binding Domain of TNFRs clonal anti-TNFR1 (sc-7895) and -TNFR2 (sc-7862) antibodies were obtained from Santa Cruz Biotechnology Inc. and used to detect the amounts of TNFR1 and -2 proteins in U-937 and HeLa cells, respectively. Cell surface receptors for different ligands were detected following the method described previously [11]. Study of Molecular Docking—The x-ray structure of the extracellular domain of TNFR1 (PDB code: 1TNR) [13] in complex with TNF is available. The x-ray structure of the TNFR1-TNF complex (1TNR) [13] was downloaded from the PDB data base. From this complex, the TNFR1 structure was extracted and used for docking with azadirachtin. The docking solution (TNFR-azadirachtin complex) with the best GOLD score was considered for further analysis

RESULTS

Azadirachtin Blocks the TNF

DISCUSSION

ADDITIONS AND CORRECTIONS