title: Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells.
abstract: OBJECTIVE
  Butyrate is produced by some types of anaerobic periodontal bacteria. Millimolar concentrations of butyrate are found in mature dental plaque from periodontitis patients. Although butyrate reportedly has a variety of effects in many mammalian cells, its effect on gingival epithelial cells is not well known. In this study, we investigated the effect of butyrate on gingival epithelial Ca9-22 cell death.
  
  
  METHODS
  Death of Ca9-22 cells was assessed after treating the cells with or without butyrate. A SYTOX Green dye, which exhibits strong green fluorescence once it enters dead cells through ruptured cell membranes, was used for cell death detection. Phosphatidylserine redistribution was measured using fluorescein isothiocyanate-labeled annexin V. The activity of caspase-3 was measured as the amount of cleaved substrate peptide. Anti-apoptotic bcl-2 mRNA expression was measured using real-time RT-PCR. Western blotting and fluoromicroscopic analysis with anti-microtubule-associated protein 1 light chain 3 (LC3) antibodies were performed for detection of autophagy.
  
  
  RESULTS
  Stimulation with millimolar concentrations of butyrate for 48 h induced Ca9-22 cell death. The stimulation also caused increased caspase-3 activity, phosphatidylserine redistribution and bcl-2 down-regulation, suggesting butyrate-induced apoptosis. However, the pan-caspase inhibitor, Z-VAD-FMK, did not inhibit cell death completely. This implies the existence of other types of cell death. In addition, markers of autophagy, namely, the conversion of LC3-I to LC3-II and increased LC3 accumulation, were observed. Moreover, inhibition of autophagy by 3-methyladenine suppressed the butyrate-induced cell death, suggesting that butyrate could induce cell death through autophagy.
  
  
  CONCLUSIONS
  These data suggest that butyrate induces apoptosis and autophagic cell death.
authors.affiliation: Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan. tsuda-h@dent.nihon-u.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Tsuda
authors.forename: K
authors.initials: K
authors.lastname: Ochiai
authors.forename: N
authors.initials: N
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Otsuka
mesh_terms: D017304:Annexin A5; D017209:Apoptosis; D001343:Autophagy; D001421:Bacteria, Anaerobic / Q000378:metabolism; D002087:Butyrates / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D015536:Down-Regulation; D004847:Epithelial Cells / Q000187:drug effects*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D009930:Organic Chemicals; D010718:Phosphatidylserines / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051025:bcl-Associated Death Protein / Q000096:biosynthesis
chemical_list: D017304:Annexin A5; D002087:Butyrates; D061945:Caspase Inhibitors; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D010718:Phosphatidylserines; C402795:SYTOX Green; D051025:bcl-Associated Death Protein; D053148:Caspase 3; D016650:Fluorescein-5-isothiocyanate