title: SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas.
abstract: BACKGROUND
  High toxicity, morbidity and secondary malignancy render chemotherapy of neuroblastoma inefficient, prompting the search for novel compounds. Nanovesicles offer great promise in imaging and treatment of cancer. SapC-DOPS, a stable nanovesicle formed from the lysosomal protein saposin C and dioleoylphosphatidylserine possess strong affinity for abundantly exposed surface phosphatidylserine on cancer cells. Here, we show that SapC-DOPS effectively targets and suppresses neuroblastoma growth and elucidate the molecular mechanism of SapC-DOPS action in neuroblastoma in vitro.
  
  
  METHODS
  In vivo targeting of neuroblastoma was assessed in xenograft mice injected intravenously with fluorescently-labeled SapC-DOPS. Xenografted tumors were also used to demonstrate its therapeutic efficacy. Apoptosis induction in vivo was evaluated in tumor sections using the TUNEL assay. The mechanisms underlying the induction of apoptosis by SapC-DOPS were addressed through measurements of cell viability, mitochondrial membrane potential (ΔΨM), flow cytometric DNA fragmentation assays and by immunoblot analysis of second mitochondria-derived activator of caspases (Smac), Bax, Cytochrome c (Cyto c) and Caspase-3 in the cytosol or in mitochondrial fractions of cultured neuroblastoma cells.
  
  
  RESULTS
  SapC-DOPS showed specific targeting and prevented the growth of human neuroblastoma xenografts in mice. In neuroblastoma cells in vitro, apoptosis occurred via a series of steps that included: (1) loss of ΔΨM and increased mitochondrial superoxide formation; (2) cytosolic release of Smac, Cyto c, AIF; and (3) mitochondrial translocation and polymerization of Bax. ShRNA-mediated Smac knockdown and V5 peptide-mediated Bax inhibition decreased cytosolic Smac and Cyto c release along with caspase activation and abrogated apoptosis, indicating that Smac and Bax are critical mediators of SapC-DOPS action. Similarly, pretreatment with the mitochondria-stabilizing agent bongkrekic acid decreased apoptosis indicating that loss of ΔΨM is critical for SapC-DOPS activity. Apoptosis induction was not critically dependent on reactive oxygen species (ROS) production and Cyclophilin D, since pretreatment with N-acetyl cysteine and cyclosporine A, respectively, did not prevent Smac or Cyto c release.
  
  
  CONCLUSIONS
  Taken together, our results indicate that SapC-DOPS acts through a mitochondria-mediated pathway accompanied by an early release of Smac and Bax. Specific tumor-targeting capacity and anticancer efficacy of SapC-DOPS supports its potential as a dual imaging and therapeutic agent in neuroblastoma therapy.
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. khan_mahaboob@yahoo.com.
authors.forename: Mahaboob K
authors.initials: MK
authors.lastname: Sulaiman
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. chuza@ucmail.uc.edu.|Divison of Human Genetics, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA. chuza@ucmail.uc.edu.
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Chu
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. blancovb@ucmail.uc.edu.
authors.forename: Victor M
authors.initials: VM
authors.lastname: Blanco
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. valabsa@ucmail.uc.edu.
authors.forename: Subrahmanya D
authors.initials: SD
authors.lastname: Vallabhapurapu
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. francors@ucmail.uc.edu.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Franco
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. xiaoyang.qi@uc.edu.|Divison of Human Genetics, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA. xiaoyang.qi@uc.edu.
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Qi
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000378:metabolism; D016572:Cyclosporine / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008819:Mice, Nude; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D053758:Nanoparticles / Q000008:administration & dosage; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D049231:Saposins / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D000081406:Peptidyl-Prolyl Isomerase F; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; C000631446:PPIF protein, mouse; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D049231:Saposins; D051028:bcl-2-Associated X Protein; C042511:1,2-dioleoylphosphatidylserine; D016572:Cyclosporine; D045304:Cytochromes c; D053148:Caspase 3; D021983:Cyclophilins