title: The In Vitro Effect of Acidic-Pepsin on Nuclear Factor KappaB Activation and Its Related Oncogenic Effect on Normal Human Hypopharyngeal Cells.
abstract: BACKGROUND
  Extra-esophageal carcinogenesis has been widely discussed in relation to the chronic effects of laryngopharyngeal reflux and most prominently with pepsin historically central to this discussion. With refluxate known to include gastric (pepsin) and duodenal (bile) fluids, we recently demonstrated the mechanistic role of NF-κB in mediating the preneoplastic effects of acidic-bile. However, the role of pepsin in promoting hypopharyngeal premalignant events remains historically unclear. Here, we investigate the in vitro effect of acidic-pepsin on the NF-κB oncogenic pathway to better define its potential role in hypopharyngeal neoplasia.
  
  
  METHODS
  Human hypopharyngeal primary cells (HHPC) and keratinocytes (HHK) were repetitively exposed to physiologic pepsin concentrations (0.1 mg/ml) at pH 4.0, 5.0 and 7.0. Cellular localization of phospho-NF-κB and bcl-2 was determined using immunofluorescence and western blotting. NF-κB transcriptional activity was tested by luc reporter and qPCR. Analysis of DNA content of pepsin treated HHK and HHPC was performed using Fluorescence-activated-cell sorting assay. To explore a possible dose related effect, pepsin concentration was reduced from 0.1 to 0.05 and 0.01 mg/ml.
  
  
  RESULTS
  At physiologic concentration, acidic-pepsin (0.1 mg/ml at pH 4.0) is lethal to most normal hypopharyngeal cells. However, in surviving cells, no NF-κB transcriptional activity is noted. Acidic-pepsin fails to activate the NF-κB or bcl-2, TNF-α, EGFR, STAT3, and wnt5α but increases the Tp53 mRNAs, in both HHPC and HHK. Weakly acidic-pepsin (pH 5.0) and neutral-pepsin (pH 7.0) induce mild activation of NF-κB with increase in TNF-α mRNAs, without oncogenic transcriptional activity. Lower concentrations of pepsin at varying pH do not produce NF-κB activity or transcriptional activation of the analyzed genes.
  
  
  CONCLUSIONS
  Our findings in vitro do not support the role of acidic-pepsin in NF-κB related hypopharyngeal carcinogenesis.
authors.affiliation: The Yale Larynx laboratory, Department of Surgery, Yale School of Medicine, New Haven, CT, United States of America.
authors.forename: Clarence T
authors.initials: CT
authors.lastname: Sasaki
authors.affiliation: The Yale Larynx laboratory, Department of Surgery, Yale School of Medicine, New Haven, CT, United States of America.
authors.forename: Julia
authors.initials: J
authors.lastname: Toman
authors.affiliation: The Yale Larynx laboratory, Department of Surgery, Yale School of Medicine, New Haven, CT, United States of America.
authors.forename: Dimitra
authors.initials: D
authors.lastname: Vageli
mesh_terms: D001646:Bile / Q000378:metabolism; D063646:Carcinogenesis; D002277:Carcinoma / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D004938:Esophageal Neoplasms / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007013:Hypopharynx / Q000166:cytology / Q000378:metabolism*; D007249:Inflammation; D015603:Keratinocytes / Q000166:cytology; D008856:Microscopy, Fluorescence; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D011230:Precancerous Conditions / Q000235:genetics; D015398:Signal Transduction; D051996:Transcription Factor RelA / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: C498143:RELA protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D010434:Pepsin A