Reactive oxygen species dependent phosphorylation of the liver kinase B1/AMP activated protein kinase/ acetyl-CoA carboxylase signaling is critically involved in apoptotic effect of lambertianic acid in hepatocellular carcinoma cells

Abstract

Though lambertianic acid (LA) is reported to have hypolipidemic activity in liver, its underlying anticancer mechanism is poorly understood so far. Thus, in the present study, apoptotic mechanism of LA was elucidated in HepG2 and SK-Hep1 hepatocellular carcinoma (HCC) cells. Here LA increased cytotoxicity, sub-G1 population and Annexin V/PI positive cells in two HCC cells. Also, LA cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP), activated phosphorylation of liver kinase B1 (LKB1)/AMP activated protein kinase (AMPK)/ acetyl-CoA carboxylase (ACC) pathway and also suppressed antiapoptotic proteins such as phosphorylation of Akt/ mammalian target of rapamycin (mTOR) and the expression of B cell lymphoma-2 (Bcl-2)/ B-cell lymphoma-extra large (Bcl-xL) and cyclooxygenase-2 (COX-2) in two HCC cells. Furthermore, LA generated reactive oxygen species (ROS) in HepG2 cells and AMPK inhibitor compound C or ROS inhibitor N-acetyl-L-cysteine (NAC) blocked the apoptotic ability of LA to cleave PARP or increase sub G1 population in HepG2 cells. Consistently, cleavages of PARP and caspase-3 were induced by LA only in AMPK^+/+ MEF cells, but not in AMPK^-/- MEF cells. Also, immunoprecipitation (IP) revealed that phosphorylation of LKB1/AMPK through their binding was enhanced in LA treated HepG2 cells. Overall, these findings suggest that ROS dependent phosphorylation of LKB1/AMPK/ACC signaling is critically involved in LA induced apoptosis in HCCs.