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- ItemPeriodic Exposure of Keratinocytes to Cold Physical Plasma: An In Vitro Model for Redox-Related Diseases of the Skin(London: Hindawi, 2016) Schmidt, Anke; von Woedtke, Thomas; Bekeschus, SanderOxidative stress illustrates an imbalance between radical formation and removal. Frequent redox stress is critically involved in many human pathologies including cancer, psoriasis, and chronic wounds. However, reactive species pursue a dual role being involved in signaling on the one hand and oxidative damage on the other. Using a HaCaT keratinocyte cell culture model, we investigated redox regulation and inflammation to periodic, low-dose oxidative stress after two, six, eight, ten, and twelve weeks. Chronic redox stress was generated by recurrent incubation with cold physical plasma-treated cell culture medium. Using transcriptome microarray technology, we identified both acute ROS-stress responses as well as numerous adaptions after several weeks of redox challenge. We determined a differential expression (2-fold, FDR < 0.01, p < 0.05) of 260 genes that function in inflammation and redox homeostasis, such as cytokines (e.g., IL-6, IL-8, and IL-10), growth factors (e.g., CSF2, FGF, and IGF-2), and antioxidant enzymes (e.g., HMOX, NQO1, GPX, and PRDX). Apoptotic signaling was affected rather modestly, especially in p53 downstream targets (e.g., BCL2, BBC3, and GADD45). Strikingly, the cell-protective heat shock protein HSP27 was strongly upregulated (p < 0.001). These results suggested cellular adaptions to frequent redox stress and may help to better understand the inflammatory responses in redox-related diseases.
- ItemNrf2 signaling and inflammation are key events in physical plasma-spurred wound healing(Wyoming, NSW : Ivyspring, 2019) Schmidt, Anke; Woedtke, Thomas, von; Vollmar, Brigitte; Hasse, Sybille; Bekeschus, SanderWound healing is strongly associated with the presence of a balanced content of reactive species in which oxygen-dependent, redox-sensitive signaling represents an essential step in the healing cascade. Numerous studies have demonstrated that cold physical plasma supports wound healing due to its ability to deliver a beneficial mixture of reactive species directly to the cells. Methods: We described a preclinical proof-of-principle-concept of cold plasma use in a dermal, full-thickness wound model in immunocompetent SKH1 mice. Quantitative PCR, Western blot analysis, immunohistochemistry and immunofluorescence were perfomed to evaluate the expression and cellular translocation of essential targets of Nrf2 and p53 signaling as well as immunomodulatory and angiogenetic factors. Apoptosis and proliferation were detected using TUNEL assay and Ki67 staining, respectively. Cytokine levels in serum were measured using bead-based multiplex cytokine analysis. Epidermal keratinocytes and dermal fibroblasts were isolated from mouse skin to perform functional knockdown experiments. Intravital fluorescence analysis was used to illustrate and quantified microvascular features. Results: Plasma exerted significant effects on wound healing in mice, including the promotion of granulation and reepithelialization as a consequence of the migration of skin cells, the balance of antioxidant and inflammatory response, and the early induction of macrophage and neutrophil recruitment to the wound sites. Moreover, through an early and local plasma-induced p53 inhibition with a concomitant stimulation of proliferation, the upregulation of angiogenetic factors, and an increased outgrowth of new vessels, our findings explain why dermal skin repair is accelerated. The cellular redox homeostasis was maintained and cells were defended from damage by a strong modulation of the nuclear E2-related factor (Nrf2) pathway and redox-sensitive p53 signaling. Conclusions: Although acute wound healing is non-problematic, the pathways highlighted that mainly the activation of Nrf2 signaling is a promising strategy for the clinical use of cold plasma in chronic wound healing.
- 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.
- 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.
- ItemRedox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma(Austin, Tex. : Landes Bioscience, 2015) Bekeschus, Sander; Schmidt, Anke; Bethge, Lydia; Masur, Kai; von Woedtke, Thomas; Hasse, Sybille; Wende, KristianIn plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.
- ItemRedox for Repair: Cold Physical Plasmas and Nrf2 Signaling Promoting Wound Healing(Basel : MDPI, 2018-10-19) Schmidt, Anke; Bekeschus, SanderChronic wounds and ulcers are major public health threats. Being a substantial burden for patients and health care systems alike, better understanding of wound pathophysiology and new avenues in the therapy of chronic wounds are urgently needed. Cold physical plasmas are particularly effective in promoting wound closure, irrespective of its etiology. These partially ionized gases deliver a therapeutic cocktail of reactive oxygen and nitrogen species safely at body temperature and without genotoxic side effects. This field of plasma medicine reanimates the idea of redox repair in physiological healing. This review compiles previous findings of plasma effects in wound healing. It discusses new links between plasma treatment of cells and tissues, and the perception and intracellular translation of plasma-derived reactive species via redox signaling pathways. Specifically, (i) molecular switches governing redox-mediated tissue response; (ii) the activation of the nuclear E2-related factor (Nrf2) signaling, together with antioxidative and immunomodulatory responses; and (iii) the stabilization of the scaffolding function and actin network in dermal fibroblasts are emphasized in the light of wound healing.
- ItemCold Physical Plasma Modulates p53 and Mitogen-Activated Protein Kinase Signaling in Keratinocytes(London: Hindawi, 2019) Schmidt, Anke; Bekeschus, Sander; Jarick, Katja; Hasse, Sybille; von Woedtke, Thomas; Wende, KristianSmall reactive oxygen and nitrogen species (ROS/RNS) driven signaling plays a significant role in wound healing processes by controlling cell functionality and wound phase transitions. The application of cold atmospheric pressure plasma (CAP), a partially ionized gas expelling a variety of ROS and RNS, was shown to be effective in chronic wound management and contrastingly also in malignant diseases. The underlying molecular mechanisms are not well understood but redox signaling events are involved. As a central player, the cellular tumor antigen p53 governs regulatory networks controlling proliferation, death, or metabolism, all of which are grossly modulated by anti- and prooxidant signals. Using a human skin cell model, a transient phosphorylation and nuclear translocation of p53, preceded by the phosphorylation of upstream serine- (ATM) and serine/threonine-protein kinase (ATR), was detected after CAP treatment. Results indicate that ATM acts as a direct redox sensor without relevant contribution of phosphorylation of the histone A2X, a marker of DNA damage. Downstream events are the activation of checkpoint kinases Chk1/2 and several mitogen-activated (MAP) kinases. Subsequently, the expression of MAP kinase signaling effectors (e.g., heat shock protein Hsp27), epithelium derived growth factors, and cytokines (Interleukins 6 + 8) was increased. A number of p53 downstream effectors pointed at a decrease of cell growth due to DNA repair processes. In summary, CAP treatment led to an activation of cell repair and defense mechanisms including a modulation of paracrine inflammatory signals emphasizing the role of prooxidant species in CAP-related cell signaling. © 2019 Anke Schmidt et al.
- ItemRisk assessment of kINPen plasma treatment of four human pancreatic cancer cell lines with respect to metastasis(Basel : MDPI AG, 2019) Bekeschus, Sander; Freund, Eric; Spadola, Chiara; Privat-Maldonado, Angela; Hackbarth, Christine; Bogaerts, Annemie; Schmidt, Anke; Wende, Kristian; Weltmann, Klaus-Dieter; Woedtke, Thomas von; Heidecke, Claus-Dieter; Partecke, Lars-Ivo; Käding, AndréCold physical plasma has limited tumor growth in many preclinical models and is, therefore, suggested as a putative therapeutic option against cancer. Yet, studies investigating the cells’ metastatic behavior following plasma treatment are scarce, although being of prime importance to evaluate the safety of this technology. Therefore, we investigated four human pancreatic cancer cell lines for their metastatic behavior in vitro and in chicken embryos (in ovo). Pancreatic cancer was chosen as it is particularly metastatic to the peritoneum and systemically, which is most predictive for outcome. In vitro, treatment with the kINPen plasma jet reduced pancreatic cancer cell activity and viability, along with unchanged or decreased motility. Additionally, the expression of adhesion markers relevant for metastasis was down-regulated, except for increased CD49d. Analysis of 3D tumor spheroid outgrowth showed a lack of plasma-spurred metastatic behavior. Finally, analysis of tumor tissue grown on chicken embryos validated the absence of an increase of metabolically active cells physically or chemically detached with plasma treatment. We conclude that plasma treatment is a safe and promising therapeutic option and that it does not promote metastatic behavior in pancreatic cancer cells in vitro and in ovo. © 2019 by the authors.
- 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.
- ItemHmox1 Upregulation Is a Mutual Marker in Human Tumor Cells Exposed to Physical Plasma-Derived Oxidants(Basel : MDPI, 2018-10-27) Bekeschus, Sander; Freund, Eric; Wende, Kristian; Gandhirajan, Rajesh; Schmidt, AnkeIncreasing numbers of cancer deaths worldwide demand for new treatment avenues. Cold physical plasma is a partially ionized gas expelling a variety of reactive oxygen and nitrogen species, which can be harnesses therapeutically. Plasmas and plasma-treated liquids have antitumor properties in vitro and in vivo. Yet, global response signatures to plasma treatment have not yet been identified. To this end, we screened eight human cancer cell lines to investigate effects of low-dose, tumor-static plasma-treated medium (PTM) on cellular activity, immune-modulatory properties, and transcriptional levels of 22 redox-related genes. With PTM, a moderate reduction of metabolic activity and modest modulation of chemokine/cytokine pattern and markers of immunogenic cell death was observed. Strikingly, the Nuclear factor (erythroid-derived 2)-like 2 (nrf2) target heme oxygenase 1 (hmox1) was upregulated in all cell lines 4 h post PTM-treatment. nrf2 was not changed, but its baseline expression inversely and significantly correlated with hmox1 expression after exposure to PTM. Besides awarding hmox1 a central role with plasma-derived oxidants, we present a transcriptional redox map of 22 targets and chemokine/cytokine secretion map of 13 targets across eight different human tumor cell lines of four tumor entities at baseline activity that are useful for future studies in this field.