Gambogic acid covalently modifies IκB kinase-β subunit to mediate suppression of lipopolysaccharide-induced activation of NF-κB in macrophages

GA (gambogic acid) is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi with a long history of use as a complementary and alternative medicine. The antitumour activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the antitumour activity of GA is mediated by its ligation of TfR1 (transferrin receptor-1). Since the cellular expression of TfR1 is down-regulated by LPS (lipopolysaccharide), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of NF-κB (nuclear factor κB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-κB-luciferase reporter and gel-shift analyses revealed that GA strongly blocked the activation of NF-κB induced by LPS, whereas 9,10-dihydro-GA, which lacks the reactive α,β-unsaturated carbonyl group, was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. In vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKβ [IκB (inhibitory κB) kinase-β], a key kinase of the NF-κB signalling axis, was covalently modified by GA at Cys-179, causing significant inhibition of its kinase activity. Taken together, these results demonstrate the potent anti-inflammatory activity of GA.