title: Abrogation of Autophagy by Chloroquine Alone or in Combination with mTOR Inhibitors Induces Apoptosis in Neuroendocrine Tumor Cells.
abstract: Everolimus (RAD001), an mTORC1 inhibitor, demonstrated promising, but limited, anticancer effects in neuroendocrine tumors (NETs). Torin1 (a global mTOR inhibitor) and NVP-BEZ235 (a PI3K/mTOR inhibitor) seem to be more effective than RAD001. Autophagy, a degradation pathway that may promote tumor growth, is regulated by mTOR; mTOR inhibition results in stimulation of autophagy. Chloroquine (CQ) inhibits autophagy.
  
  
  
  To explore the effect of CQ alone or in combination with RAD001, Torin1 or NVP-BEZ235 on autophagy and on NET cell viability, proliferation and apoptosis.
  
  
  
  The NET cell line BON1 was treated with CQ with or without different mTOR inhibitors. siRNA against ATG5/7 was used to genetically inhibit autophagy. Cellular viability was examined by XTT, proliferation by Ki-67 staining and cell cycles by flow cytometry. Apoptosis was analyzed by Western blotting for cleaved caspase 3 and staining for annexin V; autophagy was evaluated by Western blotting and immunostaining for LC3.
  
  
  
  RAD001, Torin1, NVP-BEZ235 and CQ all decreased BON1 cell viability. The effect of RAD001 was smaller than that of the other mTOR inhibitors or CQ. Torin1 and NVP-BEZ235 markedly inhibited cell proliferation, without inducing apoptosis. CQ similarly decreased cell proliferation, while robustly increasing apoptosis. Treatment with Torin1 or NVP-BEZ235 together with CQ was additive on viability, without increasing CQ-induced apoptosis. Inhibition of autophagy by ATG5/7 knockdown increased apoptosis in the presence or absence of mTOR inhibitors, mimicking the CQ effects.
  
  
  
  CQ inhibits NET growth by inducing apoptosis and by inhibiting cell proliferation, probably via inhibition of autophagy. CQ may potentiate the antitumor effect of mTOR inhibitors.
authors.affiliation: Neuroendocrine Tumor Unit, Endocrinology and Metabolism Service, Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: Shani
authors.initials: S
authors.lastname: Avniel-Polak
authors.forename: Gil
authors.initials: G
authors.lastname: Leibowitz
authors.forename: Yael
authors.initials: Y
authors.lastname: Riahi
authors.forename: Benjamin
authors.initials: B
authors.lastname: Glaser
authors.forename: David J
authors.initials: DJ
authors.lastname: Gross
authors.forename: Simona
authors.initials: S
authors.lastname: Grozinsky-Glasberg
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D002738:Chloroquine / Q000494:pharmacology*; D000068338:Everolimus / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D019394:Ki-67 Antigen / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D018358:Neuroendocrine Tumors / Q000473:pathology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011804:Quinolines / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D013997:Time Factors
chemical_list: D000970:Antineoplastic Agents; D007093:Imidazoles; D019394:Ki-67 Antigen; C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D011804:Quinolines; D002738:Chloroquine; D000068338:Everolimus; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; C531198:dactolisib