Adenosine Activates AMPK to Phosphorylate Bcl-XL Responsible for Mitochondrial Damage and DIABLO Release in HuH-7 Cells

摘要

Background/Aims Accumulating evidence has pointed to AMP-activated protein kinase (AMPK) as an inducer of apoptosis in a variety of cancer cells. The present study aimed at understanding AMPK signals for adenosine-induced HuH-7 cell apoptosis. Methods Cell viability, AMPK activity, mitochondrial membrane potentials, phosphorylation of Bcl-XsubL/sub, iin situ/i DIABLO mobilizations, and caspase-3 activity were monitored in HuH-7 cells. Plasmid DNAs for DIABLO-GFP, mutant Bcl-XsubL/sub, dominant negative mutant AMPKα2 and the siRNAs to silence the AMPKα1 or AMPKα2 targeted gene were constructed and transfected. Results Adenosine or the AMPK activator AICAR induced apoptosis in HuH-7 cells, and no synergistic effect was obtained with co-treatment. Adenosine activated AMPK, to phosphorylate Bcl-XsubL/sub. Adenosine or AICAR disrupted mitochondrial membrane potentials, and the effect was inhibited by knocking-down AMPKα1 and/or AMPKα2, expressing dominant negative mutant AMPKα2 or mutant Bcl-XsubL/sub lacking Ser/Thr phosphorylation sites. AICAR stimulated DIABLO release from the mitochondria, and the release was suppressed by expressing the mutant Bcl-XsubL/sub. AICAR activated caspase-3, which was also inhibited by expressing the mutant Bcl-XsubL/sub. Conclusion Adenosine activates AMPK, to disrupt mitochondrial membrane potentials through Bcl-XsubL/sub phosphorylation, allowing DIABLO release from the mitochondria, as a factor for caspase-3 activation to induce HuH-7 cell apoptosis.