The Mechanism Of Garcinol-induced P300/FOXP3Degradation And Its Application In Treatment For Cancer

Post-translational modifications, such as phosphorylation, acetylation, methylationand ubiquitination have been shown to occur on many transcriptional factors andaffect transcriptional activity by regulating protein stability, subcellular localization,oligomerization, or DNA binding. FOXP3lysine ε-acetylation is an important post-translational modification that regulates FOXP3expression levels and functions inTreg cells. At least two histone acetyltransferases （HAT）, including Tat-interactiveprotein （Tip60, also known as KAT5） and p300, promote FOXP3acetylation.Acetylated forms of FOXP3appear to be more resistant to poly-ubiquitination andproteasomal degradation, and move from the nucleoplasm to the chromatin.Garcinol, a polyisoprenylated benzophenone derivative from the fruit rind ofGarcinia indica, has been claimed to represent a natural inhibitor of p300.Garcinolinhibits p300auto-acetylation as well as p300-mediated p53（also known as TP53）acetylation.Our recent studies have defined a complex set of histone acetyltransferaseinteractions which can lead to enhanced or repressed changes in FOXP3function. Wehave explored the use of a natural p300inhibitor, Garcinol, as a tool to understandmechanisms by which p300regulates FOXP3acetylation. In the presence of Garcinol,p300appears to become disassociated from the FOXP3complex and undergoeslysosome-dependent degradation. As a consequence of p300’s physical absence,FOXP3becomes less acetylated and eventually degraded, a process that cannot berescued by the proteasome inhibitor MG132. p300plays a complex role in FOXP3acetylation, as it could also acetylate a subset of four Lys residues that repressivelyregulate total FOXP3acetylation.In vitro Treg suppression assay,Mouse CD4^-CD25^highTreg and CD4^-CD25-CD45RB^highT cells（representing effector T cell, Teff） were isolated fromC57BL/6mice. Teffand Treg cells were mixed at different ratios. We next examinedwhether Garcinol affected Treg suppressive function. Treg cells inhibited theproliferation of Teffcells in a dose-dependent manner.Garcinol acts as a degradationdevice to reduce the suppressive activity of Treg cells.In MTT assay demonstrated that7.16.4could inhibit H2N113in adose-dependent manner.We did note that Garcinol slightly inhibited the proliferationof H2N113.However, Garcinol was not able to enhance the activity of7.16.4in this invitro assay. Studying mouse tumor models,we show that the high dose7.16.4（9mg/kg）treatment alone significantly reduced the tumor growth. The growth of tumorappeared to be only modestly reduced by the low dose7.16.4（1.8mg/kg）treatment.While Garcinol （43.6mg/kg） had little discernible effects on the growth of tumors, thecombination of Garcinol and low dose7.16.4led to enhanced inhibition of tumorgrowth.These data indicate that Garcinol enhance the in vivo anti-tumor activity of atargeted therapeutic7.16.4monoclonal antibody in MMTV-neu transgenics implantedwith neu transformed breast tumor cells. Our studies provide the rationale formolecules that disrupt p300stability to limit Treg functions in targeted therapies forcancers.