Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells.
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.
Tsuda
H
H
Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan. tsuda-h@dent.nihon-u.ac.jp
Ochiai
K
K
Suzuki
N
N
Otsuka
K
K
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
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