Non‐thermal plasma as a simple ferroptosis inducer in cancer cells: A possible role of ferritin

To the Editor: A large number of cooperative projects are currently in progress between engineering and medical researchers worldwide. Plasma is the fourth condition of physical states out of the normal solid/liquid/gas phase and is a mixture of radicals, electrons, cations, anions and light. Aurora and thunder are typical plasma in nature, and plasma is even more abundant in space as the sun itself is plasma. However, they are usually found at extremely high temperature. Non-thermal plasma (NTP), called also low-temperature plasma, cold plasma or non-equilibrium atmospheric plasma, only became available as a novel engineering device in the late 1990s, and emits plasma of near bodytemperature. Since then, many researchers have been studying its possible applications to medicine, including its use for wounds in the battlefields and as a cancer therapy. “Plasma medical science innovation” has been a national project in Japan, and was designated as an innovative research area by the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government. Masaru Hori (Plasma Nanotechnology Research Center, Nagoya University), has been the head of this research group from 2012 to the present. Nagoya University has produced a machine that emits NTP of the highest electron density (1.6 10 cm ) as far as we know. We have thus far characterized the biological effects of NTP and found that direct NTP exposure can confer oxidative stress of specified intensity precisely to the designated location. We have applied most of the possible pre-existing methods, including measurement of conjugated diens, thiobarbituric acid-reactive substances, 4-hydroxy-2-nonenal-modified proteins, acroleinmodified proteins, 8-hydroxy-2’-deoxyguanosine and also cyclobutane pyrimidine dimers, which were all significantly and dose-dependently increased after exposure to NTP. Electron spin resonance (ESR) using spin trapping agents, which is a physical method to identify free radicals, showed that direct NTP exposure mainly produced hydroxyl radicals. Hydroxyl radicals are chemically produced through the Fenton reaction both in vitro and in vivo. In most cases, iron works as a catalyst: Fe(II)þH2O2!Fe(III)þ •OHþOH-. Iron is the most abundant heavy metal in humans and 2.5–4g is present in the whole body. Whereas its deficiency causes anemia, its excess is a risk for cancer. We demonstrated that local iron excess is the major pathogenesis of asbestos-induced mesothelical carcinogenesis. Thus, we have used malignant mesothelioma cells for the NTP exposure, which were more sensitive than fibroblasts. Furthermore, mesothelioma cell death was nonapoptotic, proportionally iron-dependent and with increased catalytic Fe(II) in the cytoplasm, which thus falls into the category of ferroptosis. Ferroptosis is a recently defined type of regulated necrosis, where iron-dependent lipid peroxidation in phospholipids and the antagonizing cystine/glutamate antiporter and glutathione peroxidase 4 are the key regulators. Intriguingly, a redox-inactive iron chelator to cover all the 6 ligands of iron can prevent ferroptosis whereas Fe(II)rich cancer cells are specifically killed by NTP. Iron is also a nutrient for cells. No species on earth can live without iron. Cancer cells accumulate iron through transferrin receptor and divalent metal transporter 1 (SLC11A2) for their proliferation, where catalytic Fe(II) is generally increased in cancer cells in comparison to nontumorous cells. Simultaneously cancer cells are under oxidative stress in comparison to their counterparts. These could be the Achilles’ heel of cancer cells. Fe(III) is almost insoluble (10 M) to water at neutral pH. Ferritin, a 440 kDa protein consisting of 24 units, is localized in the cytosol as iron storage and one molecule can harbor 4,500 Fe(III) molecules as safe non-catalytic condition. In previous experiments on the use of NTP as cancer therapy, several different molecular mechanisms have been suggested, including apoptosis in glioblastoma and lysosome genesis/autophagy in mesothelioma. We hypothesized that NTP-induced destruction of the shell of ferritin and simultaneous reduction from Fe(III) to Fe(II) may be one of the novel molecular mechanisms. Here we have performed an in vitro experiment, using ESR spin trapping, to evaluate whether NTP can reduce Fe (III) within ferritin to release Fe(II), by the use of Fenton reaction (Fig. 1). We observed the generation of hydroxyl radicals with the combined use of ferritin (from equine spleen, Sigma-Aldrich, St Louis, MO, USA), H2O2 and NTP exposure in the presence of a spin trapping agent, 5,5dimethyl-1-pyrroline N-oxide (DMPO; Labotec, Tokyo, Japan), indicating that stored Fe(III) in ferritin was indeed reduced to free catalytic Fe(II) after release from ferritin. Therefore, in irradiated cells with NTP, certain portion of Fe (III), stored in ferritin, may be reduced to free catalytic Fe(II), initiating Fenton reaction to cause oxidative cell death, which further requires demonstration in vivo in the near future. In summary, it is highly possible that we have now obtained a handy method (NTP) to induce ferroptosis in