title: Role of AMP-activated protein kinase in cross-talk between apoptosis and autophagy in human colon cancer.
abstract: Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay between autophagy and apoptosis.
authors.affiliation: Department of Surgery, University of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA 15213, USA.
authors.forename: X
authors.initials: X
authors.lastname: Song
authors.affiliation: Department of Surgery, University of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA 15213, USA.
authors.forename: S-Y
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Surgery, University of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA 15213, USA.
authors.forename: D
authors.initials: D
authors.lastname: Tang
authors.affiliation: Department of Surgery, University of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA 15213, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Bartlett
authors.affiliation: Protein Metabolism Medical Research Center and Department of Biomedical Science, College of Medicine, Seoul National University, Seoul 110-799, South Korea.
authors.forename: Y T
authors.initials: YT
authors.lastname: Kwon
authors.affiliation: 1] Department of Surgery, University of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA 15213, USA [2] Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Y J
authors.initials: YJ
authors.lastname: Lee
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D000071186:Beclin-1; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008558:Melphalan / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D020123:Sirolimus / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D001897:Boronic Acids; D008565:Membrane Proteins; D011719:Pyrazines; D010768:Phosphoserine; D000069286:Bortezomib; D055372:AMP-Activated Protein Kinases; D008558:Melphalan; D020123:Sirolimus