title: Electrolytically generated acid functional water inhibits NF-κB activity by attenuating nuclear-cytoplasmic shuttling of p65 and p50 subunits.
abstract: BACKGROUND
  Human β-defensin 2 (hBD2) gene expression is dependent on nuclear factor kappa B (NF-κB) activity. We have previously demonstrated that electrolytically generated acid functional water (FW) induces the expression of hBD2 in the human oral squamous cell carcinoma (OSCC) cell line Ca9-22. However, the induction was not dependent on NF-κB activity; in fact, FW inhibited NF-κB activity. Therefore, we hypothesized that FW might reduce spontaneous interleukin 8 (IL-8) secretion by Ca9-22 cells, which is heavily dependent on NF-κB activity. This study aimed at demonstrating the inhibitory effect of FW on NF-κB activity.
  
  
  METHODS
  Ca9-22 cells were incubated with FW, and spontaneous IL-8 secretion was observed by enzyme-linked immunosorbent assay. Luciferase assay was performed using the 5'-untranslated region of the IL-8 gene. The steps of NF-κB activation blocked by FW were evaluated by localization of the NF-κB subunits p65 and p50 by immunofluorescence staining. Western blotting was further performed to confirm the changes in NF-κB subunit localization.
  
  
  RESULTS
  The Ca9-22 cells spontaneously secreted IL-8, which was rapidly and drastically inhibited by FW treatment. The luciferase assay demonstrated the inhibitory action of FW, which was diminished by deletion of the NF-κB binding site from this construct. FW treatment altered the distribution of both the p65 and p50 subunits. P65, which was localized in the nucleus during the resting state, moved to the cytoplasm after FW treatment, whereas, p50, localized in the cytoplasm during the resting state, moved to the nucleus subsequent to FW treatment.
  
  
  CONCLUSIONS
  The results from this study indicate that FW might inhibit spontaneous IL-8 secretion by redistribution of the NF-κB subunits within the cells.
authors.affiliation: a Department of Pathobiology , Nihon University School of Dentistry , Tokyo , Japan .|b Division of Immunology and Pathobiology , Nihon University School of Dentistry , Tokyo , Japan .
authors.forename: Takahiro
authors.initials: T
authors.lastname: Gojoubori
authors.affiliation: c Division of Applied Oral Sciences , Nihon University Graduate School of Dentistry , Tokyo , Japan .
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Ota
authors.affiliation: c Division of Applied Oral Sciences , Nihon University Graduate School of Dentistry , Tokyo , Japan .
authors.forename: Masafumi
authors.initials: M
authors.lastname: Kusunoki
authors.affiliation: d Division of Oral Structural and Functional Biology , Nihon University Graduate School of Dentistry , Tokyo , Japan , and.
authors.forename: Yukina
authors.initials: Y
authors.lastname: Nishio
authors.affiliation: c Division of Applied Oral Sciences , Nihon University Graduate School of Dentistry , Tokyo , Japan .
authors.forename: Kensuke
authors.initials: K
authors.lastname: Nishio
authors.affiliation: e Division of Oral Health Sciences , Nihon University Graduate School of Dentistry , Tokyo , Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Iwasa
authors.affiliation: a Department of Pathobiology , Nihon University School of Dentistry , Tokyo , Japan .
authors.forename: Yasuhide
authors.initials: Y
authors.lastname: Kaneko
authors.affiliation: a Department of Pathobiology , Nihon University School of Dentistry , Tokyo , Japan .|b Division of Immunology and Pathobiology , Nihon University School of Dentistry , Tokyo , Japan .
authors.forename: Masatake
authors.initials: M
authors.lastname: Asano
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D004572:Electrolysis; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D051996:Transcription Factor RelA / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014867:Water / Q000494:pharmacology*
chemical_list: D016209:Interleukin-8; D016328:NF-kappa B; D021122:Protein Subunits; D051996:Transcription Factor RelA; D014867:Water
keywords: Electrolytically generated acid functional water; IL-8; NF-κB; oral squamous cell carcinoma