Abstract
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 (H2O2) in cultured oral SCC cells were measured. Treatment with safingol increased intracellular H2O2 levels but not extracellular H2O2 levels, indicating the production of H2O2. The cell killing effect of safingol and H2O2 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 H2O2 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 H2O2 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 H2O2 and safingol. These results suggest that H2O2 is involved in the endo G-mediated apoptosis of oral SCC cells by safingol.
Highlights
Apoptosis, the best-described type of programmed cell death, is characterized by cell membrane blebbing, a reduction in cellular volume, the activation of caspases, chromatin condensation and nuclear fragmentation [1,2]
We investigated the possible involvement of H2O2 as a reactive oxygen species (ROS) in endonuclease G (endo G)-mediated apoptosis of oral squamous cell carcinoma (SCC) cells treated with safingol
SAS cells were incubated with hydrogen peroxide (H2O2) or safingol, and extracellular and intracellular levels of H2O2 were measured using an assay kit for measuring H2O2 concentration [19,20]
Summary
The best-described type of programmed cell death, is characterized by cell membrane blebbing, a reduction in cellular volume, the activation of caspases, chromatin condensation and nuclear fragmentation [1,2]. Endo G is an endonuclease that is released from the mitochondrial intermembrane space and translocates to the cell nucleus to induce DNA fragmentation in a caspase-independent manner [4,5,6]. Our previous studies indicated that safingol induced apoptosis of oral squamous cell carcinoma (SCC) cells, accompanied by the nuclear translocation of endo G from mitochondria in a caspase 3-independet manner, using DNA fragmentation assay, flow cytometric analysis and immunostaining [17], but upstream mediators remain largely unknown. We investigated the possible involvement of H2O2 as a ROS in endo G-mediated apoptosis of oral SCC cells treated with safingol
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