title: Gambogic acid covalently modifies IkappaB kinase-beta subunit to mediate suppression of lipopolysaccharide-induced activation of NF-kappaB in macrophages.
abstract: 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-kappaB (nuclear factor kappaB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-kappaB-luciferase reporter and gel-shift analyses revealed that GA strongly blocked the activation of NF-kappaB induced by LPS, whereas 9,10-dihydro-GA, which lacks the reactive alpha,beta-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 IKKbeta [IkappaB (inhibitory kappaB) kinase-beta], a key kinase of the NF-kappaB 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.
authors.affiliation: Center for Molecular Immunology and Infectious Disease, Department of Veterinary and Biomedical Sciences, 115 Henning Building, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Umamaheshwari D
authors.initials: UD
authors.lastname: Palempalli
authors.forename: Ujjawal
authors.initials: U
authors.lastname: Gandhi
authors.forename: Parisa
authors.initials: P
authors.lastname: Kalantari
authors.forename: Hema
authors.initials: H
authors.lastname: Vunta
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Arner
authors.forename: Vivek
authors.initials: V
authors.lastname: Narayan
authors.forename: Anand
authors.initials: A
authors.lastname: Ravindran
authors.forename: K Sandeep
authors.initials: KS
authors.lastname: Prabhu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry*; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects*; D051996:Transcription Factor RelA / Q000378:metabolism*; D044004:Xanthones / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: D008070:Lipopolysaccharides; D051996:Transcription Factor RelA; D044004:Xanthones; C052659:gambogic acid; D051550:I-kappa B Kinase