title: PM02734 (elisidepsin) induces caspase-independent cell death associated with features of autophagy, inhibition of the Akt/mTOR signaling pathway, and activation of death-associated protein kinase.
abstract: OBJECTIVE
  PM02734 (elisidepsin) is a synthetic marine-derived cyclic peptide of the kahalalide family currently in phase II clinical development. The mechanisms of cell death induced by PM02734 remain unknown.
  
  
  METHODS
  Human non-small-cell lung cancer (NSCLC) cell lines H322 and A549 were used to evaluate PM02734-induced cytotoxicity, apoptosis, and autophagy, as well as effects on cell death-related signaling pathways.
  
  
  RESULTS
  PM02734 at clinically achievable concentrations (0.5-1 μmol/L) was cytotoxic to H322 and A549 cells but did not cause nuclear fragmentation, PARP cleavage, or caspase activation, suggesting that classical apoptosis is not its main mechanism of cell death. In contrast, PM02734-induced cell death was associated with several characteristics of autophagy, including an increase in acidic vesicular organelle content, levels of GFP-LC3-positive puncta, elevation of the levels of Atg-5/12 and LC3-II, and an associated compromise of the autophagic flux resulting in increased number of autophagosomes and/or autolysosomes. Cotreatment with 3-methyladenine (3-MA) and downregulation of Atg-5 gene expression by siRNA partially inhibited PM02734-induced cell death. PM02734 caused inhibition of Akt/mTOR signaling pathways and cotreatment with the Akt inhibitor wortmannin or with the mTOR inhibitor rapamycin led to a significant increase in PM02734-induced cell death. Furthermore, PM02734 caused the activation of death-associated protein kinase (DAPK) by dephosphorylation at Ser308, and downregulation of DAPK expression with siRNA caused also a partial but significant reduction of PM02734-induced cell death. In vivo, PM02734 significantly inhibited subcutaneous A549 tumor growth in nude mice (P < 0.05) in association with induction of autophagy.
  
  
  CONCLUSIONS
  Our data indicate that PM02734 causes cell death by a complex mechanism that involves increased autophagosome content, due for the most part to impairment of autophagic flux, inhibition of the Akt/mTOR pathway, and activation of DAPK. This unique mechanism of action justifies the continued development of this agent for the treatment of NSCLC.
authors.affiliation: Division of Medical Oncology, Department of Surgery, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Yi-He
authors.initials: YH
authors.lastname: Ling
authors.forename: Miguel
authors.initials: M
authors.lastname: Aracil
authors.forename: Yiyu
authors.initials: Y
authors.lastname: Zou
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Yuan
authors.forename: Bo
authors.initials: B
authors.lastname: Lu
authors.forename: José
authors.initials: J
authors.lastname: Jimeno
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Cuervo
authors.forename: Roman
authors.initials: R
authors.lastname: Perez-Soler
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects*; D000071187:Autophagy-Related Protein 5; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D064550:Death-Associated Protein Kinases; D047630:Depsipeptides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: C502778:ATG5 protein, human; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; D047630:Depsipeptides; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; C522358:elisidepsin; D049452:Green Fluorescent Proteins; C025946:3-methyladenine; C546842:MTOR protein, human; D064550:Death-Associated Protein Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020169:Caspases; D000225:Adenine