title: c-Jun N-terminal kinase activation by nitrobenzoxadiazoles leads to late-stage autophagy inhibition.
abstract: BACKGROUND
  Nitrobenzoxadiazole derivatives (NBDs), including NBDHEX and the recently developed MC3181, are promising anticancer agents able to target glutathione transferase and inhibit both its catalytic activity and ability to sequester TNF-receptor associated factor 2 (TRAF2) and c-Jun N-terminal kinase (JNK). NBDs have been shown to impair the growth and survival of a broad-spectrum of tumor types, in vitro and in vivo. Herein, we evaluated the effects of the new compound MC3181 on U-2OS osteosarcoma cells and investigated the impact of both NBDHEX and MC3181 on autophagy.
  
  
  METHODS
  Cell viability was evaluated by sulforhodamine B assay. The dissociation of the TRAF2-GSTP1-1 complex was detected by proximity ligation assay, while the phospho-activation of JNK was assessed by western blotting. The effects of NBDs on autophagy were evaluated by GFP-LC3 puncta formation, western blotting for LC3-II and p62, and LC3 turnover assay in the presence of bafilomycin A1. The role of JNK in the reduction of autophagic flux caused by NBDs was investigated using JNK1 shRNA-transfected cells. Fluorogenic caspase activity assay and flow cytometric analysis of DNA content were used to determine the cytotoxic effects of NBDs on JNK1-silenced cells.
  
  
  RESULTS
  Similar to NBDHEX, MC3181 reduced viability and activated TRAF2/JNK signaling in U-2OS cells. Moreover, NBDs induced the accumulation of autophagic vesicles and LC3-II while reducing both basal and nutritional stress-induced autophagic flux. Furthermore, increased levels of both LC3-II and the autophagy selective substrate p62 were observed in different tumor cell lines treated with NBDs, the concurrent increase of these markers being consistent with an impairment of autophagosome clearance. Autophagy inhibition by NBDs required JNK activity: NBDs caused autophagy inhibition and caspase-3 activation in JNK-positive U-2OS, but no autophagic flux inhibition or caspase-3 activation in JNK-silenced cells.
  
  
  CONCLUSIONS
  Our demonstration that NBDs can act as late-phase autophagy inhibitors opens new opportunities to fully exploit their therapeutic potential. This may not rely solely on their effectiveness in inducing cell cycle arrest and apoptosis, but also on their ability to weaken the capacity of tumor cells to endure stress conditions via autophagy. In addition, this study provides evidence that JNK can participate in impairing autophagy.
authors: Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy. camilla.palumbo@uniroma2.it.
authors: Camilla
authors: C
authors: Palumbo
authors: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy. anastasia.deluca@gmail.com.
authors: Anastasia
authors: A
authors: De Luca
authors: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy. nicola.rosato@uniroma2.it.|The NAST Centre for Nanoscience and Nanotechnology and Innovative Instrumentation, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy. nicola.rosato@uniroma2.it.
authors: Nicola
authors: N
authors: Rosato
authors: Department of Drug Chemistry and Technologies, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy. Mariantonietta.Forgione@iit.it.|Center for Life Nano Science@Sapienza, Italian Institute of Technology, Viale Regina Elena 291, 00161, Rome, Italy. Mariantonietta.Forgione@iit.it.
authors: Mariantonietta
authors: M
authors: Forgione
authors: Department of Drug Chemistry and Technologies, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy. danterotili@libero.it.
authors: Dante
authors: D
authors: Rotili
authors: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy. caccuri@uniroma2.it.|The NAST Centre for Nanoscience and Nanotechnology and Innovative Instrumentation, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy. caccuri@uniroma2.it.
authors: Anna Maria
authors: AM
authors: Caccuri
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology; D010069:Oxadiazoles / Q000494:pharmacology*; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013312:Stress, Physiological / Q000187:drug effects; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism
chemical_list: C528479:6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D010069:Oxadiazoles; C118585:P62 protein, human; D016601:RNA-Binding Proteins; D047992:TNF Receptor-Associated Factor 2; D048031:JNK Mitogen-Activated Protein Kinases; D053148:Caspase 3