The Mechanism Of Action Of 18?-glycyrrhetinic Acid Oxadadiazole Analogues 10d And 10e In Human Leukemia Cells

18?-glycyrrhetinic acid(GA)is a naturally occurring oleanane-type pentacyclic triterpenoid isolated from the plant(Glycyrrhiza glabra)with moderate antitumor activities.To improve the antitumor activity,we modified the structure such as A ring,C ring,and C-30 of GA,and got the compound Methyl 2-cyano-3,12-dioxo-18?-olean-1,9(11)-dien-30-oate(CDODO-Me,10d),which has a strong ability of inducting apoptosis and binding to intracellular reduced glutathione(GSH).To avoid the GSH binding ability,methyl 2-cyano-3-oxo-18?-olean-1,9(11),12-trien-30-oate(COOTO,10e)with a change at c-ring has been synthesized,it has the decreased ability of binding to GSH and maintaining the ability of apoptosis induction.We compared the explored the anti-leukemia effect of both compounds and the mechanisms of action.Both 10d and 10e induced apoptosis in HL-60 cells at similar molecular concentrations with the activation of caspase-8 and caspase-9,associated with the down-regulation of anti-apoptotic protein c-Flip,Mcl-1 and the up-regulation of pro-apoptotic protein Noxa.Jurkat cells without expression of caspase-8 were less sensitive to 10e,suggesting that the activations of caspase-8 plays roles in the apoptosis induction due to the down-regulation of c-Flip.We investigated the mechanisms of 10d resulting in down-regulation of c-Flip and Mcl-1 as well as up-regulation of Noxa.The pan-caspase inhibitor Z-VAD-FMK blocked the reduction of Mcl-1,but not of c-Flip reduction or Noxa induction in 10e-treated cells.The reduction of c-Flip and Mcl-1 by 10e was blocked by proteasomal inhibitor MG 132.These data suggest that 10e regulates those proteins through different mechanisms.The decrease of Mcl-1 was mediated by inhibition of AKT/mTOR pathway.The upregulation of Noxa through a CHOP-mediated pathway,which led to Bak activation through inactivating Mcl-1.Silencing of Noxa or Bak significantly attenuated 10e-induced apoptosis.Ku70 stabilizes c-Flip and inactivates Bax by forming complexes which is controlled by acetylation.10e decreased the level of HDAC3 protein,increased the acetylation of Ku70,and dissociated c-Flip and Bax from Ku70,leading to c-Flip degradation but Bax activation.We revealed a new mechanism of 10e to induce apoptosis by interrupting the complexes of Ku70 with Bax and c-Flip through degrading HDAC3 protein.Our work reveal a new direction for target-driven modification of glycyrrhetinic acid and other triterpenoids.Considering the multiple oncogene induction and combined therapy is required for leukemia,We tested the combined effects of Bcl-2 inhibitor ABT-199 or the proteasome inhibitor Bortezomib with 10e,and that the synergistic effect of inducting apoptosis was found.Biotin-labeled 10d and 10e,10e containing one unsaturated ketone group has poor protein binding ability that 10d which has two unsaturated ketone groups,and 10e has the ability of apoptosis induction,thus A ring is the mainly reason of inducting apoptosis.