title: Antioxidant inhibition of steady-state reactive oxygen species and cell growth in neuroblastoma.
abstract: BACKGROUND
  Reactive oxygen species (ROS) contribute to adult tumorigenesis; however, their roles in pediatric solid tumors are unknown. Here, we sought to define the steady-state ROS levels in neuroblastoma and to examine whether aggressive cellular behavior, which may predict treatment failure, is regulated by ROS.
  
  
  METHODS
  Neuroblastoma sections were assessed for 4-hydroxynonenal (4-HNE), a marker of intracellular lipid peroxidation and a byproduct of increased levels of ROS. Human neuroblastoma cell lines, MYCN-amplified BE(2)-C and MYCN-nonamplified SK-N-SH, were examined in our study. Superoxide and hydroperoxide oxidation products were detected by staining for dihydroethidium (DHE) and 5, 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (CDCFH2), using the oxidation-insensitive analog CDCF as a negative control. Cells were treated with N-acetylcysteine (NAC; 10 mmol/L) daily for 5 days and analyzed.
  
  
  RESULTS
  Greater expression of 4-HNE was observed in undifferentiated tumor sections as compared with the more differentiated tumors. Interestingly, increased levels of ROS were detected in MYCN-amplified BE(2)-C cells. Moreover, gastrin-releasing peptide receptor-induced ROS production stimulated upregulation of the hypoxia inducible factor (HIF)-1α/vascular endothelial growth factor (VEGF) pathway and an increase in cell growth. Antioxidant NAC decreased HIF-1α/VEGF expression and inhibited BE(2)-C cell growth.
  
  
  CONCLUSIONS
  We report a novel observation that shifting the redox balance toward greater ROS levels results in a more aggressive neuroblastoma phenotype. Our data suggest that ROS play a critical role in refractory neuroblastoma.
authors.affiliation: Department of Radiation Oncology, Northwestern School of Medicine, Chicago, IL.
authors.forename: Yueming
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN; Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN.
authors.forename: Pritha
authors.initials: P
authors.lastname: Paul
authors.affiliation: Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN; Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN.
authors.forename: Sora
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Craig
authors.affiliation: Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rellinger
authors.affiliation: Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN.
authors.forename: Jingbo
authors.initials: J
authors.lastname: Qiao
authors.affiliation: Department of Radiation Oncology, Northwestern School of Medicine, Chicago, IL.
authors.forename: David R
authors.initials: DR
authors.lastname: Gius
authors.affiliation: Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN; Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN. Electronic address: dai.chung@vanderbilt.edu.
authors.forename: Dai H
authors.initials: DH
authors.lastname: Chung
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D009447:Neuroblastoma / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000975:Antioxidants; D014408:Biomarkers, Tumor; D017382:Reactive Oxygen Species; D000111:Acetylcysteine