title: Effect of dual inhibition of apoptosis and autophagy in prostate cancer.
abstract: OBJECTIVE
  Targeting multiple anti-apoptotic proteins is now possible with the small molecule BH3 domain mimetics such as ABT-737. Given recent studies demonstrating that autophagy is a resistance mechanism to multiple therapeutic agents in the setting of apoptotic inhibition, we hypothesized that hydroxychloroquine (HCQ), an anti-malarial drug that inhibits autophagy, will increase cytotoxicity of ABT-737.
  
  
  METHODS
  Cytotoxicity of ABT-737 and HCQ was assessed in vitro in PC-3 and LNCaP cells, and in vivo in a xenograft mouse model. The role of autophagy as a resistance mechanism was assessed by siRNA knockdown of the essential autophagy gene beclin1. ROS was measured by flow cytometry, and mitophagy assessed by the mCherry-Parkin reporter.
  
  
  RESULTS
  Induction of autophagy by ABT-737 was a mechanism of resistance in prostate cancer cell lines. Therapeutic inhibition of autophagy with HCQ increased cytotoxicity of ABT-737 both in vitro and in vivo. ABT-737 induced LC-3 and decreased p62 expression by immunoblot in cell lines and by immunohistochemistry in tumors in vivo. Assessment of ROS and mitochondria demonstrated that ROS production by ABT-737 and HCQ was a mechanism of cytotoxicity.
  
  
  CONCLUSIONS
  We demonstrated that autophagy inhibition with HCQ enhances ABT-737 cytotoxicity in vitro and in vivo, that LC-3 and p62 represent assessable markers in human tissue for future clinical trials, and that ROS induction is a mechanism of cytotoxicity. These results support a new paradigm of dual targeting of apoptosis and autophagy in future clinical studies.
authors.affiliation: The Cancer Institute of New Jersey, New Brunswick, New Jersey, USA.
authors.forename: Ahamed
authors.initials: A
authors.lastname: Saleem
authors.forename: Dmitri
authors.initials: D
authors.lastname: Dvorzhinski
authors.forename: Urmila
authors.initials: U
authors.lastname: Santanam
authors.forename: Robin
authors.initials: R
authors.lastname: Mathew
authors.forename: Kevin
authors.initials: K
authors.lastname: Bray
authors.forename: Mark
authors.initials: M
authors.lastname: Stein
authors.forename: Eileen
authors.initials: E
authors.lastname: White
authors.forename: Robert S
authors.initials: RS
authors.lastname: DiPaola
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D001713:Biphenyl Compounds / Q000008:administration & dosage; D045744:Cell Line, Tumor; D004359:Drug Therapy, Combination; D006801:Humans; D006886:Hydroxychloroquine / Q000008:administration & dosage; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009596:Nitrophenols / Q000008:administration & dosage; D010879:Piperazines / Q000008:administration & dosage; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D013449:Sulfonamides / Q000008:administration & dosage; D016896:Treatment Outcome; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: C501332:ABT-737; D001713:Biphenyl Compounds; D009596:Nitrophenols; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D006886:Hydroxychloroquine