title: Intermittent hypoxia effect on osteoclastogenesis stimulated by neuroblastoma cells.
abstract: BACKGROUND
  Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions.
  
  
  METHODS
  We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone.
  
  
  RESULTS
  Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells.
  
  
  CONCLUSIONS
  Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.
authors.affiliation: Department of Cancer Biology and pharmacology, University of Illinois College of Medicine, Peoria, Illinois, United States of America.
authors.forename: Vasantha Kumar
authors.initials: VK
authors.lastname: Bhaskara
authors.affiliation: Department of Cancer Biology and pharmacology, University of Illinois College of Medicine, Peoria, Illinois, United States of America.
authors.forename: Indra
authors.initials: I
authors.lastname: Mohanam
authors.affiliation: Department of Pathology, University of Illinois College of Medicine, Peoria, Illinois, United States of America.
authors.forename: Meena
authors.initials: M
authors.lastname: Gujrati
authors.affiliation: Department of Cancer Biology and pharmacology, University of Illinois College of Medicine, Peoria, Illinois, United States of America.
authors.forename: Sanjeeva
authors.initials: S
authors.lastname: Mohanam
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D001859:Bone Neoplasms / Q000378:metabolism / Q000556:secondary; D001862:Bone Resorption / Q000235:genetics / Q000378:metabolism*; D002450:Cell Communication; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D004195:Disease Models, Animal; D015870:Gene Expression; D064593:Heterografts; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010010:Osteoclasts / Q000378:metabolism*; D010014:Osteolysis; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D044169:Receptors, Calcium-Sensing; D042461:Vascular Endothelial Growth Factor A; D000099162:Endothelial PAS Domain-Containing Protein 1