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- 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
- ItemTumor cytotoxicity and immunogenicity of a novel V-jet neon plasma source compared to the kINPen(London : Nature Publishing Group, 2021) Miebach, Lea; Freund, Eric; Horn, Stefan; Niessner, Felix; Sagwal, Sanjeev Kumar; von Woedtke, Thomas; Emmert, Steffen; Weltmann, Klaus-Dieter; Clemen, Ramona; Schmidt, Anke; Gerling, Torsten; Bekeschus, SanderRecent research indicated the potential of cold physical plasma in cancer therapy. The plethora of plasma-derived reactive oxygen and nitrogen species (ROS/RNS) mediate diverse antitumor effects after eliciting oxidative stress in cancer cells. We aimed at exploiting this principle using a newly designed dual-jet neon plasma source (Vjet) to treat colorectal cancer cells. A treatment time-dependent ROS/RNS generation induced oxidation, growth retardation, and cell death within 3D tumor spheroids were found. In TUM-CAM, a semi in vivo model, the Vjet markedly reduced vascularized tumors' growth, but an increase of tumor cell immunogenicity or uptake by dendritic cells was not observed. By comparison, the argon-driven single jet kINPen, known to mediate anticancer effects in vitro, in vivo, and in patients, generated less ROS/RNS and terminal cell death in spheroids. In the TUM-CAM model, however, the kINPen was equivalently effective and induced a stronger expression of immunogenic cancer cell death (ICD) markers, leading to increased phagocytosis of kINPen but not Vjet plasma-treated tumor cells by dendritic cells. Moreover, the Vjet was characterized according to the requirements of the DIN-SPEC 91315. Our results highlight the plasma device-specific action on cancer cells for evaluating optimal discharges for plasma cancer treatment.
- 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.
- ItemReactive species driven oxidative modifications of peptides—Tracing physical plasma liquid chemistry(Melville, NY : American Inst. of Physics, 2021) Wenske, Sebastian; Lackmann, Jan-Wilm; Busch, Larissa Milena; Bekeschus, Sander; von Woedtke, Thomas; Wende, KristianThe effluence of physical plasma consists of a significant share of reactive species, which may interact with biomolecules and yield chemical modifications comparable to those of physiological processes, e.g., post-translational protein modifications (oxPTMs). Consequentially, the aim of this work is to understand the role of physical plasma-derived reactive species in the introduction of oxPTM-like modifications in proteins. An artificial peptide library consisting of ten peptides was screened against the impact of two plasma sources, the argon-driven MHz-jet kINPen and the helium-driven RF-jet COST-Jet. Changes in the peptide molecular structure were analyzed by liquid chromatography–mass spectrometry. The amino acids cysteine, methionine, tyrosine, and tryptophan were identified as major targets. The introduction of one, two, or three oxygen atoms was the most common modification observed. Distinct modification patterns were observed for nitration (+N + 2O–H), which occurred in kINPen only (peroxynitrite), and chlorination (+Cl–H) that was exclusive for the COST-Jet in the presence of chloride ions (atomic oxygen/hypochlorite). Predominantly for the kINPen, singlet oxygen-related modifications, e.g., cleavage of tryptophan, were observed. Oxidation, carbonylation, and double oxidations were attributed to the impact of hydroxyl radicals and atomic oxygen. Leading to a significant change in the peptide side chain, most of these oxPTM-like modifications affect the secondary structure of amino acid chains, and amino acid polarity/functionality, ultimately modifying the performance and stability of cellular proteins.
- ItemGas Plasma Technology-An Asset to Healthcare during Viral Pandemics Such as the COVID-19 Crisis?(New York, NY : IEEE, 2020) Bekeschus, Sander; Kramer, Axel; Suffredini, Elisabetta; von Woedtke, Thomas; Colombo, VittorioThe COVID-19 crisis profoundly disguised the vulnerability of human societies and healthcare systems in the situation of a pandemic. In many instances, it became evident that the quick and safe reduction of viral load and spread is the foremost principle in the successful management of such a pandemic. However, it became also clear that many of the established routines in healthcare are not always sufficient to cope with the increased demand for decontamination procedures of items, healthcare products, and even infected tissues. For the last 25 years, the use of gas plasma technology has sparked a tremendous amount of literature on its decontaminating properties, especially for heat-labile targets, such as polymers and tissues, where chemical decontamination often is not appropriate. However, while the majority of earlier work focused on bacteria, only relatively few reports are available on the inactivation of viruses. We here aim to provide a perspective for the general audience of the chances and opportunities of gas plasma technology for supporting healthcare during viral pandemics such as the COVID-19 crisis. This includes possible real-world plasma applications, appropriate laboratory viral test systems, and critical points on the technical and safety requirements of gas plasmas for virus inactivation.
- ItemCombination of Gas Plasma and Radiotherapy Has Immunostimulatory Potential and Additive Toxicity in Murine Melanoma Cells In Vitro(Basel : Molecular Diversity Preservation International, 2020) Pasqual-Melo, Gabriella; Sagwal, Sanjeev Kumar; Freund, Eric; Gandhirajan, Rajesh Kumar; Frey, Benjamin; von Woedtke, Thomas; Gaipl, Udo; Bekeschus, SanderDespite continuous advances in therapy, malignant melanoma is still among the deadliest types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other way around. This was concomitant with increased levels of TNFa, IL6, and GM-CSF in supernatants. Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and radiotherapy, and translational tumor models are needed to develop this concept further. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemArgon Plasma Exposure Augments Costimulatory Ligands and Cytokine Release in Human Monocyte-Derived Dendritic Cells(Basel : Molecular Diversity Preservation International (MDPI), 2021) Bekeschus, Sander; Meyer, Dorothee; Arlt, Kevin; von Woedtke, Thomas; Miebach, Lea; Freund, Eric; Clemen, RamonaCold physical plasma is a partially ionized gas expelling many reactive oxygen and nitrogen species (ROS/RNS). Several plasma devices have been licensed for medical use in dermatology, and recent experimental studies suggest their putative role in cancer treatment. In cancer therapies with an immunological dimension, successful antigen presentation and inflammation modulation is a key hallmark to elicit antitumor immunity. Dendritic cells (DCs) are critical for this task. However, the inflammatory consequences of DCs following plasma exposure are unknown. To this end, human monocyte-derived DCs (moDCs) were expanded from isolated human primary monocytes; exposed to plasma; and their metabolic activity, surface marker expression, and cytokine profiles were analyzed. As controls, hydrogen peroxide, hypochlorous acid, and peroxynitrite were used. Among all types of ROS/RNS-mediated treatments, plasma exposure exerted the most notable increase of activation markers at 24 h such as CD25, CD40, and CD83 known to be crucial for T cell costimulation. Moreover, the treatments increased interleukin (IL)-1α, IL-6, and IL-23. Altogether, this study suggests plasma treatment augmenting costimulatory ligand and cytokine expression in human moDCs, which might exert beneficial effects in the tumor microenvironment.
- ItemEx Vivo Exposure of Human Melanoma Tissue to Cold Physical Plasma Elicits Apoptosis and Modulates Inflammation(Basel : MDPI, 2020) Bekeschus, Sander; Moritz, Juliane; Helfrich, Iris; Boeckmann, Lars; Weltmann, Klaus-Dieter; Emmert, Steffen; Metelmann, Hans-Robert; Stoffels, Ingo; von Woedtke, ThomasCutaneous melanoma is the most aggressive type of skin cancer with a not-sufficient clinical outcome. High tumor mutation rates often hamper a remedial treatment, creating the need for palliative care in many patients. To reduce pain and burden, local palliation often includes cryo-ablation, immunotherapy via injection of IL2, or electrochemotherapy. Yet, a fraction of patients and lesions do not respond to those therapies. To reach even these resistances in a redox-mediated way, we treated skin biopsies from human melanoma ex vivo with cold physical plasma (kINPen MED plasma jet). This partially ionized gas generates a potent mixture of reactive oxygen species (ROS). Physical plasmas have been shown to be potent antitumor agents in preclinical melanoma and clinical head and neck cancer research. The innovation of this technology lies in its ease-of-use without anesthesia, as the “cold” plasma temperature of the kINPen MED does not exceed 37 °C. In metastatic melanoma skin biopsies from six patients, we identified a marked increase of apoptosis with plasma treatment ex vivo. This had an impact on the chemokine/cytokine profile of the cultured biopsies, e.g., three of six patient-derived biopsy supernatants showed an apparent decrease in VEGF compared to non-plasma treated specimens. Moreover, the baseline release levels of 24 chemokines/cytokines investigated may serve as a useful tool for future research on melanoma skin biopsy treatments. Our findings suggest a clinically useful role of cold physical plasma therapy in palliation of cutaneous melanoma lesions, possibly in a combinatory setting with other immune therapies.
- ItemHyperspectral Imaging of Wounds Reveals Augmented Tissue Oxygenation following Cold Physical Plasma Treatment in Vivo(New York, NY : IEEE, 2021) Schmidt, Anke; Niesner, Felix; von Woedtke, Thomas; Bekeschus, SanderEfficient vascularization of skin tissue supports wound healing in response to injury. This includes elevated blood circulation, tissue oxygenation, and perfusion. Cold physical plasma promotes wound healing in animal models and humans. Physical plasmas are multicomponent systems that generate several physicochemical effectors, such as ions, electrons, reactive oxygen and nitrogen species, and UV radiation. However, the consequences of plasma treatment on wound oxygenation and perfusion, vital processes to promote tissue regeneration, are largely unexplored. We used a novel hyperspectral imaging (HSI) system and a murine dermal full-thickness wound model in combination with kINPen argon plasma jet treatment to address this question. Plasma treatment promoted tissue oxygenation in superficial as well as deep (6 mm) layers of wound tissue. In addition to perfusion changes, we found a wound healing stage-dependent shift of tissue hemoglobin and tissue water index during reactive species-driven wound healing. Contactless, fast monitoring of medical parameters in real-time using HSI revealed a plasma-supporting effect in wound healing together with precise information about biological surface-specific features.
- ItemThe amino acid metabolism is essential for evading physical plasma-induced tumour cell death(Edinburgh : Nature Publ. Group, 2021) Gandhirajan, Rajesh Kumar; Meyer, Dorothee; Sagwal, Sanjeev Kumar; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Bekeschus, SanderBackground: Recent studies have emphasised the important role of amino acids in cancer metabolism. Cold physical plasma is an evolving technology employed to target tumour cells by introducing reactive oxygen species (ROS). However, limited understanding is available on the role of metabolic reprogramming in tumour cells fostering or reducing plasma-induced cancer cell death. Methods: The utilisation and impact of major metabolic substrates of fatty acid, amino acid and TCA pathways were investigated in several tumour cell lines following plasma exposure by qPCR, immunoblotting and cell death analysis. Results: Metabolic substrates were utilised in Panc-1 and HeLa but not in OVCAR3 and SK-MEL-28 cells following plasma treatment. Among the key genes governing these pathways, ASCT2 and SLC3A2 were consistently upregulated in Panc-1, Miapaca2GR, HeLa and MeWo cells. siRNA-mediated knockdown of ASCT2, glutamine depletion and pharmacological inhibition with V9302 sensitised HeLa cells to the plasma-induced cell death. Exogenous supplementation of glutamine, valine or tyrosine led to improved metabolism and viability of tumour cells following plasma treatment. Conclusion: These data suggest the amino acid influx driving metabolic reprogramming in tumour cells exposed to physical plasma, governing the extent of cell death. This pathway could be targeted in combination with existing anti-tumour agents. © 2021, The Author(s).