Hydrogen Sulfide Induces Apoptosis in Human Gingival Epithelial Cell Line

Objectives:

Hydrogen sulfide (H₂S) is an important oral malodorous volatile sulfur compound also reported as having periodontal pathogenic activities. The specific aim of this study is to demonstrate another role that H₂S can play in the oral cavity. Here we show that H₂S induces apoptosis in a human gingival epithelial cell line, Ca9-22, and we determine the mechanisms underlying cellular signaling pathways.

Methods:

Samples were incubated in a special incubation system at 50ng/ml H₂S in 5% CO2 air and were analyzed after 24h and 48h. For detecting the viability and the form of cell death samples were double stained with Annexin V, and 7-AAD, and analyzed using flow cytometry. Then, we assessed the levels of key apoptotic enzymes: caspase 3, 8, 9, measuring a fluorescent marker by flow cytometry. The mitochondrial membrane potential changes were examined by fluorescence-based assay.

Results:

Necrosis was found in less than 8% of the cells during 2 days of incubation, whereas 24.9% and 35.4% of the cells were found to be apoptotic after 24h and 48h, respectively. These results were correlated with a strong activation of caspase 3 and a dramatic increment of caspase 9 levels, i.e. 45% more than in negative control. The mitochondrial membrane potential was collapsed in 30% and 50% of the H2S-treated cells after 24h and 48h incubation, respectively. Caspase 8 activity was found to have the same activity levels as negative control.

Conclusions:

The present results demonstrate the induction of apoptosis in Ca9-22, oral keratinocytes cell line, by hydrogen sulfide. The loss of mitochondrial inner membrane potential, the marked activation of both caspase 9 and caspase 3, and the absence of caspase 8 activity suggest that mitochondria-mediated caspase activation acts as the apoptotic signaling pathway by H₂S.