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- ItemToxicity and Immunogenicity in Murine Melanoma following Exposure to Physical Plasma-Derived Oxidants(Austin, Tex. : Landes Bioscience, 2017) Bekeschus, Sander; Rödder, Katrin; Fregin, Bob; Otto, Oliver; Lippert, Maxi; Weltmann, Klaus-Dieter; Wende, Kristian; Schmidt, Anke; Gandhirajan, Rajesh KumarMetastatic melanoma is an aggressive and deadly disease. Therapeutic advance has been achieved by antitumor chemo- and radiotherapy. These modalities involve the generation of reactive oxygen and nitrogen species, affecting cellular viability, migration, and immunogenicity. Such species are also created by cold physical plasma, an ionized gas capable of redox modulating cells and tissues without thermal damage. Cold plasma has been suggested for anticancer therapy. Here, melanoma cell toxicity, motility, and immunogenicity of murine metastatic melanoma cells were investigated following plasma exposure in vitro. Cells were oxidized by plasma, leading to decreased metabolic activity and cell death. Moreover, plasma decelerated melanoma cell growth, viability, and cell cycling. This was accompanied by increased cellular stiffness and upregulation of zonula occludens 1 protein in the cell membrane. Importantly, expression levels of immunogenic cell surface molecules such as major histocompatibility complex I, calreticulin, and melanocortin receptor 1 were significantly increased in response to plasma. Finally, plasma treatment significantly decreased the release of vascular endothelial growth factor, a molecule with importance in angiogenesis. Altogether, these results suggest beneficial toxicity of cold plasma in murine melanomas with a concomitant immunogenicity of potential interest in oncology.
- ItemOn a heavy path – determining cold plasma-derived short-lived species chemistry using isotopic labelling(London : RSC Publishing, 2020) Wende, Kristian; Bruno, Giuliana; Lalk, Michael; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Bekeschus, Sander; Lackmann, Jan-WilmCold atmospheric plasmas (CAPs) are promising medical tools and are currently applied in dermatology and epithelial cancers. While understanding of the biomedical effects is already substantial, knowledge on the contribution of individual ROS and RNS and the mode of activation of biochemical pathways is insufficient. Especially the formation and transport of short-lived reactive species in liquids remain elusive, a situation shared with other approaches involving redox processes such as photodynamic therapy. Here, the contribution of plasma-generated reactive oxygen species (ROS) in plasma liquid chemistry was determined by labeling these via admixing heavy oxygen 18O2 to the feed gas or by using heavy water H218O as a solvent for the bait molecule. The inclusion of heavy or light oxygen atoms by the labeled ROS into the different cysteine products was determined by mass spectrometry. While products like cysteine sulfonic acid incorporated nearly exclusively gas phase-derived oxygen species (atomic oxygen and/or singlet oxygen), a significant contribution of liquid phase-derived species (OH radicals) was observed for cysteine-S-sulfonate. The role, origin, and reaction mechanisms of short-lived species, namely hydroxyl radicals, singlet oxygen, and atomic oxygen, are discussed. Interactions of these species both with the target cysteine molecule as well as the interphase and the liquid bulk are taken into consideration to shed light onto several reaction pathways resulting in observed isotopic oxygen incorporation. These studies give valuable insight into underlying plasma–liquid interaction processes and are a first step to understand these interaction processes between the gas and liquid phase on a molecular level.
- ItemROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy(London: Hindawi, 2019) Privat-Maldonado, Angela; Schmidt, Anke; Lin, Abraham; Weltmann, Klaus-Dieter; Wende, Kristian; Bogaerts, Annemie; Bekeschus, SanderPhysical plasmas generate unique mixes of reactive oxygen and nitrogen species (RONS or ROS). Only a bit more than a decade ago, these plasmas, operating at body temperature, started to be considered for medical therapy with considerably little mechanistic redox chemistry or biomedical research existing on that topic at that time. Today, a vast body of evidence is available on physical plasma-derived ROS, from their spatiotemporal resolution in the plasma gas phase to sophisticated chemical and biochemical analysis of these species once dissolved in liquids. Data from in silico analysis dissected potential reaction pathways of plasma-derived reactive species with biological membranes, and in vitro and in vivo experiments in cell and animal disease models identified molecular mechanisms and potential therapeutic benefits of physical plasmas. In 2013, the first medical plasma systems entered the European market as class IIa devices and have proven to be a valuable resource in dermatology, especially for supporting the healing of chronic wounds. The first results in cancer patients treated with plasma are promising, too. Due to the many potentials of this blooming new field ahead, there is a need to highlight the main concepts distilled from plasma research in chemistry and biology that serve as a mechanistic link between plasma physics (how and which plasma-derived ROS are produced) and therapy (what is the medical benefit). This inevitably puts cellular membranes in focus, as these are the natural interphase between ROS produced by plasmas and translation of their chemical reactivity into distinct biological responses. © 2019 Angela Privat-Maldonado et al.
- ItemDevelopment of an electrochemical sensor for in-situ monitoring of reactive species produced by cold physical plasma(Amsterdam [u.a.] : Elsevier Science, 2021) Nasri, Zahra; Bruno, Giuliana; Bekeschus, Sander; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Wende, KristianThe extent of clinical applications of oxidative stress-based therapies such as photodynamic therapy (PDT) or respiratory chain disruptors are increasing rapidly, with cold physical plasma (CPP) emerging as a further option. According to the current knowledge, the biological effects of CPP base on reactive oxygen and nitrogen species (RONS) relevant in cell signaling. To monitor the safety and the biological impact of the CPP, determining the local generation of RONS in the same environment in which they are going to be applied is desirable. Here, for the first time, the development of an electrochemical sensor for the simple, quick, and parallel determination of plasma-generated reactive species is described. The proposed sensor consists of a toluidine blue redox system that is covalently attached to a gold electrode surface. By recording chronoamperometry at different potentials, it is possible to follow the in-situ production of the main long-lived reactive oxygen and nitrogen species like hydrogen peroxide, nitrite, hypochlorite, and chloramine with time. The applicability of this electrochemical sensor for the in-situ assessment of reactive species in redox-based therapies is demonstrated by the precise analysis of hydrogen peroxide dynamics in the presence of blood cancer cells.
- ItemNon-steroidal anti-inflammatory drugs: recent advances in the use of synthetic COX-2 inhibitors(Cambridge : Royal Society of Chemistry, 2022) Ahmadi, Mohsen; Bekeschus, Sander; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Wende, KristianCyclooxygenase (COX) enzymes comprise COX-1 and COX-2 isoforms and are responsible for prostaglandin production. Prostaglandins have critical roles in the inflammation pathway and must be controlled by administration of selective nonsteroidal anti-inflammatory drugs (NSAIDs). Selective COX-2 inhibitors have been among the most used NSAIDs during the ongoing coronavirus 2019 pandemic because they reduce pain and protect against inflammation-related diseases. In this framework, the mechanism of action of both COX isoforms (particularly COX-2) as inflammation mediators must be reviewed. Moreover, proinflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-6, IL-1β, and IL-8 must be highlighted due to their major participation in upregulation of the inflammatory reaction. Structural and functional analyses of selective COX-2 inhibitors within the active-site cavity of COXs could enable introduction of lead structures with higher selectivity and potency against inflammation with fewer adverse effects. This review focuses on the biological activity of recently discovered synthetic COX-2, dual COX-2/lipoxygenase, and COX-2/soluble epoxide hydrolase hybrid inhibitors based primarily on the active motifs of related US Food and Drug Administration-approved drugs. These new agents could provide several advantages with regard to anti-inflammatory activity, gastrointestinal protection, and a safer profile compared with those of the NSAIDs celecoxib, valdecoxib, and rofecoxib.
- ItemA Comparison of Floating-Electrode DBD and kINPen Jet: Plasma Parameters to Achieve Similar Growth Reduction in Colon Cancer Cells Under Standardized Conditions(Dordrecht : Springer Science + Business Media B.V., 2017-9-6) Bekeschus, Sander; Lin, Abraham; Fridman, Alexander; Wende, Kristian; Weltmann, Klaus-Dieter; Miller, VandanaA comparative study of two plasma sources (floating-electrode dielectric barrier discharge, DBD, Drexel University; atmospheric pressure argon plasma jet, kINPen, INP Greifswald) on cancer cell toxicity was performed. Cell culture protocols, cytotoxicity assays, and procedures for assessment of hydrogen peroxide (H2O2) were standardized between both labs. The inhibitory concentration 50 (IC50) and its corresponding H2O2 deposition was determined for both devices. For the DBD, IC50 and H2O2 generation were largely dependent on the total energy input but not pulsing frequency, treatment time, or total number of cells. DBD cytotoxicity could not be replicated by addition of H2O2 alone and was inhibited by larger amounts of liquid present during the treatment. Jet plasma toxicity depended on peroxide generation as well as total cell number and amount of liquid. Thus, the amount of liquid present during plasma treatment in vitro is key in attenuating short-lived species or other physical effects from plasmas. These in vitro results suggest a role of liquids in or on tissues during plasma treatment in a clinical setting. Additionally, we provide a platform for correlation between different plasma sources for a predefined cellular response.
- ItemOxygen atoms are critical in rendering THP-1 leukaemia cells susceptible to cold physical plasma-induced apoptosis([London] : Macmillan Publishers Limited, part of Springer Nature, 2017-6-5) Bekeschus, Sander; Wende, Kristian; Hefny, Mohamed Mokhtar; Rödder, Katrin; Jablonowski, Helena; Schmidt, Anke; Woedtke, Thomas von; Weltmann, Klaus-Dieter; Benedikt, JanCold physical plasma has been suggested as a powerful new tool in oncology. However, some cancer cells such as THP-1 leukaemia cells have been shown to be resistant towards plasma-induced cell death, thereby serving as a good model for optimizing plasmas in order to foster pro-apoptotic anticancer effects. A helium/oxygen radio frequency driven atmospheric plasma profoundly induced apoptosis in THP-1 cells whereas helium, humidified helium, and humidified helium/oxygen plasmas were inefficient. Hydrogen peroxide – previously shown as central plasma-derived agent – did not participate in the killing reaction but our results suggest hypochlorous acid to be responsible for the effect observed. Proteomic analysis of THP-1 cells exposed to He/O2 plasma emphasized a prominent growth retardation, cell stress, apoptosis, and a pro-immunogenic profile. Altogether, a plasma setting that inactivates previously unresponsive leukaemia cells is presented. Crucial reactive species in the plasma and liquid environment were identified and discussed, deciphering the complexity of plasma from the gas phase into the liquid down to the cellular response mechanism. These results may help tailoring plasmas for clinical applications such as oxidation-insensitive types of cancer.
- ItemElevated H2AX Phosphorylation Observed with kINPen Plasma Treatment Is Not Caused by ROS-Mediated DNA Damage but Is the Consequence of Apoptosis(London: Hindawi, 2019) Bekeschus, Sander; Schütz, Clarissa S.; Nießner, Felix; Wende, Kristian; Weltmann, Klaus-Dieter; Gelbrich, Nadine; von Woedtke, Thomas; Schmidt, Anke; Stope, Matthias B.Phosphorylated histone 2AX (γH2AX) is a long-standing marker for DNA double-strand breaks (DSBs) from ionizing radiation in the field of radiobiology. This led to the perception of γH2AX being a general marker of direct DNA damage with the treatment of other agents such as low-dose exogenous ROS that unlikely act on cellular DNA directly. Cold physical plasma confers biomedical effects majorly via release of reactive oxygen and nitrogen species (ROS). In vitro, increase of γH2AX has often been observed with plasma treatment, leading to the conclusion that DNA damage is a direct consequence of plasma exposure. However, increase in γH2AX also occurs during apoptosis, which is often observed with plasma treatment as well. Moreover, it must be questioned if plasma-derived ROS can reach into the nucleus and still be reactive enough to damage DNA directly. We investigated γH2AX induction in a lymphocyte cell line upon ROS exposure (plasma, hydrogen peroxide, or hypochlorous acid) or UV-B light. Cytotoxicity and γH2AX induction was abrogated by the use of antioxidants with all types of ROS treatment but not UV radiation. H2AX phosphorylation levels were overall independent of analyzing either all nucleated cells or segmenting γH2AX phosphorylation for each cell cycle phase. SB202190 (p38-MAPK inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited γH2AX induction upon ROS but not UV treatment. Finally, and despite γH2AX induction, UV but not plasma treatment led to significantly increased micronucleus formation, which is a functional read-out of genotoxic DNA DSBs. We conclude that plasma-mediated and low-ROS γH2AX induction depends on caspase activation and hence is not the cause but consequence of apoptosis induction. Moreover, we could not identify lasting mutagenic effects with plasma treatment despite phosphorylation of H2AX.
- ItemNonenzymatic post-translational modifications in peptides by cold plasma-derived reactive oxygen and nitrogen species(Melville, NY : AIP, 2020) Wenske, Sebastian; Lackmann, Jan-Wilm; Bekeschus, Sander; Weltmann, Klaus-Dieter; Von Woedtke, Thomas; Wende, KristianCold physical plasmas are emerging tools for wound care and cancer control that deliver reactive oxygen species (ROS) and nitrogen species (RNS). Alongside direct effects on cellular signaling processes, covalent modification of biomolecules may contribute to the observed physiological consequences. The potential of ROS/RNS generated by two different plasma sources (kINPen and COST-Jet) to introduce post-translational modifications (PTMs) in the peptides angiotensin and bradykinin was explored. While the peptide backbone was kept intact, a significant introduction of oxidative PTMs was observed. The modifications cluster at aromatic (tyrosine, histidine, and phenylalanine) and neutral amino acids (isoleucine and proline) with the introduction of one, two, or three oxygen atoms, ring cleavages of histidine and tryptophan, and nitration/nitrosylation predominantly observed. Alkaline and acidic amino acid (arginine and aspartic acid) residues showed a high resilience, indicating that local charges and the chemical environment at large modulate the attack of the electron-rich ROS/RNS. Previously published simulations, which include only OH radicals as ROS, do not match the experimental results in full, suggesting the contribution of other short-lived species, i.e., atomic oxygen, singlet oxygen, and peroxynitrite. The observed PTMs are relevant for the biological activity of peptides and proteins, changing polarity, folding, and function. In conclusion, it can be assumed that an introduction of covalent oxidative modifications at the amino acid chain level occurs during a plasma treatment. The introduced changes, in part, mimic naturally occurring patterns that can be interpreted by the cell, and subsequently, these PTMs allow for prolonged secondary effects on cell physiology. © 2020 Author(s).
- ItemSinglet-Oxygen-Induced Phospholipase A2 Inhibition: A Major Role for Interfacial Tryptophan Dioxidation(Weinheim : Wiley-VCH, 2021) Nasri, Zahra; Memari, Seyedali; Wenske, Sebastian; Clemen, Ramona; Martens, Ulrike; Delcea, Mihaela; Bekeschus, Sander; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Wende, KristianSeveral studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A2 (PLA2) activity. Therefore, the regulation of PLA2 catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA2 due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA2 inhibition. A more detailed analysis of the plasma-treated PLA2 identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N-formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation. © 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH