Involvement of Hydrogen Peroxide in Safingol-Induced Endonuclease G-Mediated Apoptosis of Squamous Cell Carcinoma Cells

Safingol, a L-threo-dihydrosphingosine, induced the nuclear translocation of a mitochondrial apoptogenic mediator—endonuclease G (endo G)—and apoptosis of human oral squamous cell carcinoma (SCC) cells. Upstream mediators remain largely unknown. The levels of hydrogen peroxide (H(2)O(2)) in cultured oral SCC cells were measured. Treatment with safingol increased intracellular H(2)O(2) levels but not extracellular H(2)O(2) levels, indicating the production of H(2)O(2). The cell killing effect of safingol and H(2)O(2) was diminished in the presence of reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). Dual staining of cells with annexin V and propidium iodide (PI) revealed that apoptotic cell death occurred by treatment with H(2)O(2) and safingol. The number of apoptotic cells was reduced in the presence of NAC. In untreated cells, endo G distributed in the cytoplasm and an association of endo G with mitochondria was observed. After treatment with H(2)O(2) and safingol, endo G was distributed to the nucleus and cytoplasm, indicating the nuclear translocation of the mitochondrial factor. NAC prevented the increase of apoptotic cells and the translocation of endo G. Knock down of endo G diminished the cell killing effect of H(2)O(2) and safingol. These results suggest that H(2)O(2) is involved in the endo G-mediated apoptosis of oral SCC cells by safingol.