title: Pepsin promotes proliferation of laryngeal and pharyngeal epithelial cells.
abstract: OBJECTIVE
  Laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngeal squamous cell carcinoma (SCC), but causality has never been proven. It is accepted that chronic reflux into the esophagus can induce metaplastic changes in esophageal mucosa with subsequent increased risk of esophageal adenocarcinoma, but no similar associations have been established for LPR and laryngopharyngeal SCC. The objective of this study was to test the hypothesis that reflux of pepsin into the laryngopharynx can promote carcinogenesis.
  
  
  METHODS
  Translational research study.
  
  
  METHODS
  Normal human laryngeal primary epithelial cell cultures and hypopharyngeal FaDu SCC cells were exposed to human pepsin and analyzed by Human Cancer PathwayFinder and miRNA Superarrays, flow cytometry, and Western blot to determine the effect of pepsin on carcinogenesis. Laryngeal biopsy specimens taken from cancer patients and normal control subjects were analyzed for the presence of pepsin by Western blot.
  
  
  RESULTS
  Microarray analysis demonstrated that pepsin significantly altered the expression of 27 genes implicated in carcinogenesis and also affected the expression of 22 microRNAs known to be altered in human head and neck cancers. Pepsin increased proliferation in both FaDu SCC cells and cultured normal laryngeal epithelial primary cells by increasing S phase distribution on flow cytometry analysis in a time- and dose-dependent manner. Furthermore, pepsin was detected in 60% (3/5) human laryngeal cancer biopsies, absent in all (0/5) normal control specimens.
  
  
  CONCLUSIONS
  These data support a role for refluxed pepsin in the promotion of epithelial proliferation and carcinogenesis of the larynx and pharynx.
authors.affiliation: Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. njohnsto@mcw.edu
authors.forename: Nikki
authors.initials: N
authors.lastname: Johnston
authors.forename: Justin C
authors.initials: JC
authors.lastname: Yan
authors.forename: Craig R
authors.initials: CR
authors.lastname: Hoekzema
authors.forename: Tina L
authors.initials: TL
authors.lastname: Samuels
authors.forename: Gary D
authors.initials: GD
authors.lastname: Stoner
authors.forename: Joel H
authors.initials: JH
authors.lastname: Blumin
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Bock
mesh_terms: D001707:Biopsy, Needle; D015153:Blotting, Western; D016022:Case-Control Studies; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007013:Hypopharynx / Q000166:cytology* / Q000187:drug effects; D007822:Laryngeal Neoplasms / Q000473:pathology*; D008297:Male; D035683:MicroRNAs / Q000032:analysis; D046228:Microarray Analysis; D010434:Pepsin A / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012016:Reference Values; D012680:Sensitivity and Specificity
chemical_list: D035683:MicroRNAs; D010434:Pepsin A