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.