Non-thermal plasma has been recognized as a promising tool across a vast variety of biomedical applications, with the potential to create novel therapeutic methods. However, the understanding of the molecular mechanisms behind non-thermal plasma cellular effects remains a significant challenge. In this study, we show how two types of different non-thermal plasmas induce cell death in mammalian cell cultures via the formation of multiple intracellular reactive oxygen/nitrogen species. Our results showed a discrepancy in the superoxide accumulation and lysosomal activity in response to air and helium plasma, suggesting that triggered signalling cascades might be grossly different between different plasmas. In addition, the effects of ozone, a considerable component of non-thermal plasma, have been simultaneously evaluated and have revealed much faster and higher cytotoxic effects. Our findings offer novel insight into plasma-induced cellular responses, and provide a basis for better controlled biomedical applications.

] Institute of Physics AS CR Prague Czech Republic [2] Institute of Experimental Medicine AS CR Prague Czech Republic

Institute of Experimental Medicine AS CR Prague Czech Republic

Institute of Macromolecular Chemistry AS CR Prague Czech Republic

Institute of Physics AS CR Prague Czech Republic

See more in PubMed

Yousfi M., Merbahi N., Pathak A. & Eichwald O. Low-temperature plasmas at atmospheric pressure: toward new pharmaceutical treatments in medicine. Fundam. Clin. Pharmacol. 28, 123–135 (2014). PubMed

De Geyter N. & Morent R. Nonthermal plasma sterilization of living and nonliving surfaces. Annu. Rev. Biomed. Eng. 14, 255–274 (2012). PubMed

Fridman G. et al. Applied plasma medicine. Plasma Process. Polym. 5, 503–533 (2008).

Levy S. B. & Marshall B. Antibacterial resistance worldwide: causes, challenges and responses. Nat. Med. 10, S122–S129 (2004). PubMed

Andersson D. I. & Hughes D. Antibiotic resistance and its cost: is it possible to reverse resistance? Nat. Rev. Microbiol. 8, 260–271 (2010). PubMed

Ermolaeva S. A. et al. Bactericidal effects of non-thermal argon plasma in vitro, in biofilms and in the animal model of infected wounds. J. Med. Microbiol. 60, 75–83 (2011). PubMed

Daeschlein G. et al. In Vitro Susceptibility of Multidrug Resistant Skin and Wound Pathogens Against Low Temperature Atmospheric Pressure Plasma Jet (APPJ) and Dielectric Barrier Discharge Plasma (DBD). Plasma Process. Polym. 11, 175–183 (2014).

Fridman A., Chirokov A. & Gutsol A. Non-thermal atmospheric pressure discharges. J. Phys. D Appl. Phys. 38, R1–R24 (2005).

Tian W. & Kushner M. J. Atmospheric pressure dielectric barrier discharges interacting with liquid covered tissue. J. Phys. D Appl. Phys. 47, 165201 (2014).

Lee D. H. et al. Suppression of scar formation in a murine burn wound model by the application of non-thermal plasma. Appl. Phys. Lett. 99, 203701 (2011).

Isbary G. et al. Cold atmospheric argon plasma treatment may accelerate wound healing in chronic wounds: Results of an open retrospective randomized controlled study in vivo. Clin. Plasma Med. 1, 25–30 (2013).

Rupf S. et al. Killing of adherent oral microbes by a non-thermal atmospheric plasma jet. J. Med. Microbiol. 59, 206–212 (2010). PubMed

Gweon B. et al. Suppression of angiogenesis by atmospheric pressure plasma in human aortic endothelial cells. Appl. Phys. Lett. 104, 133701 (2014).

Kalghatgi S. et al. Effects of non-thermal plasma on mammalian cells. PLoS One 6, e16270 (2011). PubMed PMC

Ahn H. J. et al. Atmospheric-pressure plasma jet induces apoptosis involving mitochondria via generation of free radicals. PLoS One 6, e28154 (2011). PubMed PMC

Ahn H. J. et al. Targeting cancer cells with reactive oxygen and nitrogen species generated by atmospheric-pressure air plasma. PLoS One 9, e86173 (2014). PubMed PMC

Mittler R. et al. ROS signaling: the new wave? Trends. Plant Sci. 16, 300–309 (2011). PubMed

Finkel T. Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15 (2011). PubMed PMC

Kalghatgi S., Friedman G., Fridman A. & Clyne A. M. Endothelial cell proliferation is enhanced by low dose non-thermal plasma through fibroblast growth factor-2 release. Ann. Biomed. Eng. 38, 748–757 (2010). PubMed

Gweon B. et al. Plasma effects on subcellular structures. Appl. Phys. Lett. 96, 101501 (2010).

Kosmider B., Loader J. E., Murphy R. C. & Mason R. J. Apoptosis induced by ozone and oxysterols in human alveolar epithelial cells. Free Radic. Biol. Med. 48, 1513–1524 (2010). PubMed PMC

Klestadt D., Laval-Gilly P. & Falla J. Ozone-mediated cytotoxicity after short-term exposure and its relation to the production of cellular metabolites (NO, H2O2). Cell Biol. Toxicol. 18, 259–269 (2002). PubMed

Baier M., Kandlbinder A., Golldack D. & Dietz K. J. Oxidative stress and ozone: perception, signalling and response. Plant Cell Environ. 28, 1012–1020 (2005).

Kim G. J., Kim W., Kim K. T. & Lee J. K. DNA damage and mitochondria dysfunction in cell apoptosis induced by nonthermal air plasma. Appl. Phys. Lett. 96, 021502 (2010).

Churpita O., Dejneka A., Zablotskii V., Kubinová Š. & E. S. Atmospheric plasma source for biomedical applications. Patent number, 25959 (2013).

Kim K. et al. Simple Atmospheric-Pressure Nonthermal Plasma-Jet System for Poly(dimethylsiloxane) Bonding Process. Jpn. J. Appl. Phys. 51, 06FL15 (2012).

Zimmermann J. L. et al. Test for bacterial resistance build-up against plasma treatment. New J. Phys. 14, 073037 (2012).

Park C. H. et al. Wound healing with nonthermal microplasma jets generated in arrays of hourglass microcavity devices. J. Phys. D Appl. Phys. 47, 435402 (2014).

Daeschlein G. et al. Antibacterial activity of an atmospheric pressure plasma jet against relevant wound pathogens in vitro on a simulated wound environment. Plasma Process. Polym. 7, 224–230 (2010).

Matthes R. et al. Antimicrobial efficacy of two surface barrier discharges with air plasma against in vitro biofilms. PLoS One 8, e70462 (2013). PubMed PMC

Kang S. U. et al. Nonthermal plasma induces head and neck cancer cell death: the potential involvement of mitogen-activated protein kinase-dependent mitochondrial reactive oxygen species. Cell Death Dis. 5, e1056 (2014). PubMed PMC

Panngom K. et al. Preferential killing of human lung cancer cell lines with mitochondrial dysfunction by nonthermal dielectric barrier discharge plasma. Cell Death Dis. 4, e642 (2013). PubMed PMC

Gabrielson E. W., Yu X. Y. & Spannhake E. W. Comparison of the toxic effects of hydrogen-peroxide and ozone on cultured human bronchial epithelial-cells. Environ. Health Perspect. 102, 972–974 (1994). PubMed PMC

Kuonen R. et al. Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes. Evid. Based Complement. Alternat. Med. 2013, 718105 (2013). PubMed PMC

Barth R. F. & Kaur B. Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9, RG2, F98, BT4C, RT-2 and CNS-1 gliomas. J. Neurooncol. 94, 299–312 (2009). PubMed PMC

Cheng X. Q. et al. The effect of tuning cold plasma composition on glioblastoma cell viability. PLoS One 9, e98652 (2014). PubMed PMC

Sawai H. & Domae N. Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells. Biochem. Biophys. Res. Commun. 411, 569–573 (2011). PubMed

Riccardi C. & Nicoletti I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc. 1, 1458–1461 (2006). PubMed

Temkin V. & Karin M. From death receptor to reactive oxygen species and c-Jun N-terminal protein kinase: the receptor-interacting protein 1 odyssey. Immunol. Rev. 220, 8–21 (2007). PubMed

Kroemer G. & Reed J. C. Mitochondrial control of cell death. Nat. Med. 6, 513–519 (2000). PubMed

Zhao M., Antunes F., Eaton J. W. & Brunk U. T. Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis. Eur. J. Biochem. 270, 3778–3786 (2003). PubMed

Windelborn J. A. & Lipton P. Lysosomal release of cathepsins causes ischemic damage in the rat hippocampal slice and depends on NMDA-mediated calcium influx, arachidonic acid metabolism, and free radical production. J. Neurochem. 106, 56–69 (2008). PubMed PMC

Erdal H. et al. Induction of lysosomal membrane permeabilization by compounds that activate p53-independent apoptosis. Proc. Natl. Acad. Sci. U. S. A. 102, 192–197 (2005). PubMed PMC

Lin M. T. & Beal M. F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 (2006). PubMed

Smiley S. T. et al. Intracellular Heterogeneity in Mitochondrial-Membrane Potentials Revealed by a J-Aggregate-Forming Lipophilic Cation Jc-1. Proc. Natl. Acad. Sci. U. S. A. 88, 3671–3675 (1991). PubMed PMC

Halasi M. et al. ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors. Biochem. J. 454, 201–208 (2013). PubMed PMC

Gillissen A. & Nowak D. Characterization of N-acetylcysteine and ambroxol in anti-oxidant therapy. Respir. Med. 92, 609–623 (1998). PubMed

Lunov O. et al. The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on c-Jun N-terminal kinase-mediated apoptosis in human macrophages. Biomaterials 31, 5063–5071 (2010). PubMed

Lebeer S., Vanderleyden J. & De Keersmaecker S. C. J. Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat. Rev. Microbiol. 8, 171–184 (2010). PubMed

Silhavy T. J., Kahne D. & Walker S. The Bacterial Cell Envelope. Csh. Perspect. Biol. 2, a000414 (2010). PubMed PMC

Winterbourn C. C. Reconciling the chemistry and biology of reactive oxygen species. Nat. Chem. Biol. 4, 278–286 (2008). PubMed

Boya P. Lysosomal Function and Dysfunction: Mechanism and Disease. Antioxid. Redox Sign. 17, 766–774 (2012). PubMed

Trachootham D., Alexandre J. & Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 8, 579–591 (2009). PubMed

Kim K. et al. Cellular membrane collapse by atmospheric-pressure plasma jet. Appl. Phys. Lett. 104, 013701 (2014).

Zhou J. et al. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion. Cell Res. 23, 508–523 (2013). PubMed PMC

Bjornsti M. A. & Houghton P. J. The TOR pathway: a target for cancer therapy. Nat. Rev. Cancer. 4, 335–348 (2004). PubMed

Zoncu R., Efeyan A. & Sabatini D. M. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Bio. 12, 21–35 (2011). PubMed PMC

Kang M. A., So E. Y., Simons A. L., Spitz D. R. & Ouchi T. DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway. Cell Death Dis. 3, e249 (2012). PubMed PMC

Ziegler J. F., Ziegler M. D. & Biersack J. P. SRIM - The stopping and range of ions in matter (2010). Nucl. Instrum. Meth. B 268, 1818–1823 (2010).

Butterbaugh J. W., Baston L. D. & Sawin H. H. Measurement and Analysis of Radio-Frequency Glow-Discharge Electrical-Impedance and Network Power Loss. J. Vac. Sci. Technol. A 8, 916–923 (1990).

Portable and affordable cold air plasma source with optimized bactericidal effect

Remote Actuation of Apoptosis in Liver Cancer Cells via Magneto-Mechanical Modulation of Iron Oxide Nanoparticles

Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation

Chemically different non-thermal plasmas target distinct cell death pathways

Non-thermal air plasma promotes the healing of acute skin wounds in rats