This R21 application is submitted in response to an RFA (#AT-07-005) entitled """"""""Mechanisms of Immune Modulation."""""""" The primary objective of our proposed studies is to understand the molecular mechanism of action of an important complementary medicine, gambogic acid (GA) that has a long history of medicinal use. GA is a polyprenylated xanthone, with an ?, ?-unsaturated carbonyl group, isolated from gamboges, the resin from Garcinia morella and Garcinia hanburyi. Many botanicals with such a reactive carbon center are known to possess interesting pharmacological properties. In this regard, GA, which possesses anti-tumor activity, is thought to possess anti-inflammatory activities that have so far remained untested. Due to the presence of the ?,?-unsaturated carbonyl group, GA is a potent Michael acceptor that can covalently interact with free thiol groups in proteins, including transcription factors. Our preliminary results demonstrate an inhibitory effect of GA on the bacterial lipopolysaccharide (LPS)-induced activation of nuclear factor-?B (NF-?B). NF-?B is a major transcription factor responsible for inflammation-driven disease progression. We will extend these studies to elucidate the underlying molecular mechanism of immune modulation of inflammation by GA with a specific focus on the transcription of two NF-?B target pro-inflammatory genes, cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)-?. The specific hypothesis to be tested is that GA down-regulates the transcription of COX-2 and TNF? via the formation of covalent Michael adducts with protein components of the NF-?B cascade. The hypothesis will be tested using LPS-stimulated murine bone marrow-derived macrophages and validated in an in-vivo inflammation model, in C57/BL6 mice, in two Specific Aims: 1) To examine the effect of GA on the transcriptional regulation of COX-2 and TNF?, and 2) To elucidate the molecular mechanism of attenuation of NF-?B activation and function by GA. Studies will be focused on attenuation of inflammation by GA via the transcriptional regulation of COX-2 and TNF? and will involve the use of luciferase-based reporter and chromatin immunoprecipitation assays. Furthermore, the interaction of GA with protein components of the NF-?B pathway will be assessed by using biotinylated-GA in mass spectrometric and biochemical functional studies. Our long-term objective is to enhance the understanding of the molecular mechanism of action of botanicals used in complementary and alternative medicine for their role in immune regulation of inflammation. ? ? PUBLIC HEALTH REVELANCE: Gamboges, the resin from Garcinia morella and G. hanburyi that belong to the family Clusiaceae (Guttiferae), which are native to Asia, Australia, Southern Africa, and Polynesia, are rich in a polyprenylated xanthone, gambogic acid (GA). There is a long history of medicinal use of Garcinia extracts against many ailments. Interestingly, the anti-tumor activity of GA has been well demonstrated and is thought to arise partly due to the anti-inflammatory activity associated with GA. However, the anti-inflammatory activity of GA has not been tested. We propose to study the molecular basis of the down-regulation of NF-?B-dependent expression of pro-inflammatory genes by GA in in vitro and in vivo models of inflammation. ? ? ?
