Arecoline-mediated inhibition of AMP-activated protein kinase through reactive oxygen species is required for apoptosis induction

Arecoline is the major alkaloid of areca nut (AN) and known to induce reactive oxygen species (ROS) production and apoptosis. The metabolic sensor AMP-activated protein kinase (AMPK), activated by ROS, also regulates apoptosis. This study used several types of cells as the experimental model to analyze the roles of ROS and AMPK in arecoline-induced apoptosis. We found that arecoline dose-dependently increased intracellular ROS level, and two antioxidants, N-acetyl-l-cysteine (NAC) and glutathione, attenuated arecoline-induced apoptotic cell death. Interestingly, arecoline dose- and time-dependently inhibited rather than stimulated AMPK-Thr¹⁷² phosphorylation, and both NAC and glutathione relieved this inhibition. The AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), also restored the phosphorylation level of AMPK-Thr¹⁷² and attenuated apoptotic cell death under arecoline insult. In contrast, the AMPK inhibitor, compound C, and RNA interference of AMPK expression increased the cytotoxicity of arecoline. Collectively, these results suggest that arecoline may inhibit AMPK through intracellular ROS, responsible for the execution of apoptosis.