Identification of reactive oxygen species that induce spoptosis, a novel and distinctive mode of regulated cell death

Using some solutions activated by irradiation with non-thermal atmospheric pressure plasma (NTAPP), we had discovered that a new and distinctive mode of cell death, named spoptosis, exists in cells, the induction of which involves the action of reactive oxygen species (ROS). However, it was unknown what types of ROS and how they trigger the cell death. When cells were treated with a higher dose of Ascorbic acid (AA) generating O₂^- and H₂O₂ or Antimycin A (AM) generating O₂^-, cell death occurred along with cellular shrinkage, Pdcd4 disappearance, and vesicle formation. Only in cells treated with AA, genomic DNA was digested irregularly and membrane permeability increased aberrantly. On the other hand, cells treated with a higher dose of H₂O₂ displayed cell death and cellular shrinkage but not the other events, and those treated with a lower dose of H₂O₂ displayed cell death but not the other events. Strikingly, when cells underwent double treatment with AM and H₂O₂, the events, which had not been observed by their single treatment, became compensated. All the events were suppressed with an antioxidant, confirming that they were mediated by ROS. Thus, the mode of cell death induced by AA or combination of AM and H₂O₂ was consistent with that of cell death by NTAPP-activated solutions. These results suggested that O₂^- and H₂O₂ collaboratively trigger spoptotic cell death with the associated events, and that AA and combination of AM and H₂O₂ are functionally alternative in place of NTAPP-activated solutions.