Ozone mediates tumor-selective cell death caused by air plasma-activated medium independently of NOx

Cold atmospheric plasma and plasma-treated liquids (PTLs) are emerging promising tools for tumor-targeted cancer treatment, as they preferentially injure tumor cells more than non-malignant cells. Oxidative stress is critical to the antitumor effect, but the oxidant mediating the effect is debatable. Previously, we reported that air plasma-activated medium (APAM) has tumor-selective cytotoxicity in vitro and in vivo. Moreover, an unusual mitochondrial positioning named monopolar perinuclear mitochondrial clustering (MPMC) and nuclear damage proceeds to cell death. We noticed that air plasma generation was accompanied by ozone (O3) formation, leading to suppose the possible role of O3 in the effect of APAM. In this study, we produced an O3-dissolved medium (ODM) and comparatively analyzed its biological effect with APAM. Both agents had comparable amounts of dissolved O3 (dO3), while APAM, but not ODM, contained nitrite and nitrate. Like APAM, ODM could induce apoptosis, nonapoptotic cell death, tubulin remodeling, MPMC, and nuclear shrinkage. Catalase mitigated all these events. The increases in various intracellular and mitochondrial reactive oxygen species (ROS) and lipid peroxides proceeded to cell death, and catalase also prevented them. Conversely, suppressing cellular H2O2 removal systems augmented mitochondrial ROS production and cell death. In contrast, like APAM, ODM minimally increased ROS production and MPMC in non-malignant cells. These results indicate that dO3 is a critical mediator of the actions of APAM, including tumor-selective induction of MPMC and cell death. Our findings suggest ODM could be a more chemically-defined alternative to PTLs in cancer treatment. ### Competing Interest Statement Manami Suzuki-Karasaki, Miki Suzuki-Karasaki, and Dr. Yoshihiro Suzuki-Karasaki are employees of the Non-Profit Research Institute Plasma ChemiBio Laboratory. Other authors have no conflicts of interest. The funders had no role in the study's design, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. * 7-AAD : 7-amino-actinomycin D APAM : air plasma-activated medium CAP : cold atmospheric plasma CL : cardiolipin Drp : dynamin-related protein Fer-1 : ferrostatin-1 Hoe,mn : Hoechst 33342 LPO : lipid peroxide MitoSOX : MitoSOX Red CMXRos MPMC : monopolar perinuclear mitochondrial clustering mROS : mitochondrial reactive oxygen species MTR : MitoTracker Red nROS : nuclear reactive oxygen species NAC : N-acetylcysteine NAO : 10-N-nonyl acridine orange NAC : N-acetylcysteine NC : Nocodazole OC : oral cancer OS : osteosarcoma PNMC : perinuclear mitochondrial clustering TTG : tubulin tracker green.