Bharangin, a diterpenoid quinonemethide, abolishes constitutive and inducible nuclear factor-κB (NF-κB) activation by modifying p65 on cysteine 38 residue and reducing inhibitor of nuclear factor-κB α kinase activation, leading to suppression of NF-κB-regulated gene expression and sensitization of tumor cells to chemotherapeutic agents

• Published in: Molecular pharmacology 2011-11, Vol.80 (5), p.769-781
• Main Authors: Gupta, Subash C, Kannappan, Ramaswamy, Kim, Jihye, Rahman, Ghazi M, Francis, Sajin K, Raveendran, Reshma, Nair, Mangalam S, Das, Joydip, Aggarwal, Bharat B
• Format: Article
• Language: English
• Subjects: Apoptosis; Blotting, Western; Carcinogens - pharmacology; Cell Line, Tumor; Cysteine - metabolism; Electrophoretic Mobility Shift Assay; Flavonoids - pharmacology; Gene Expression - physiology; Genes, Reporter; Humans; Immunohistochemistry; Magnetic Resonance Spectroscopy; Mass Spectrometry; NF-kappa B - chemistry; NF-kappa B - metabolism; NF-kappa B - physiology; Phosphorylation
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.111.073122

Although inflammatory pathways have been linked with various chronic diseases including cancer, identification of an agent that can suppress these pathways has therapeutic potential. Herein we describe the identification of a novel compound bharangin, a diterpenoid quinonemethide that can suppress pro-inflammatory pathways specifically. We found that bharangin suppresses nuclear factor (NF)-κB activation induced by pro-inflammatory cytokine, tumor promoter, cigarette smoke, and endotoxin. Inhibition of NF-κB activation was mediated through the suppression of phosphorylation and degradation of inhibitor of nuclear factor-κB (IκBα); inhibition of IκBα kinase activation; and suppression of p65 nuclear translocation, and phosphorylation. The diterpenoid inhibited binding of p65 to DNA. A reducing agent reversed the inhibitory effect, and mutation of the Cys(38) of p65 to serine abrogated the effect of bharangin on p65-DNA binding. Molecular docking revealed strong interaction of the ligand with the p65 via two hydrogen bonds one with Lys(37) (2.204 Å) and another with Cys(38) (2.023 Å). The inhibitory effect of bharangin on NF-κB activation was specific, inasmuch as binding of activator protein-1 and octameric transcription factor 1 to DNA was not affected. Suppression of NF-κB activation by this diterpenoid caused the down-regulation of the expression of proteins involved in tumor cell survival, proliferation, invasion, and angiogenesis, leading to potentiation of apoptosis, suppression of proliferation, and invasion of tumor cells. Furthermore, the genetic deletion of p65 and mutation of p65Cys(38) residue to Ser abolished the affect of bharangin. Overall, our results demonstrate that bharangin specifically inhibits the NF-κB activation pathway by modifying p65 and inhibiting IκBα kinase activation and potentiates apoptosis in tumor cells.