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- ItemIdentification of two kinase inhibitors with synergistic toxicity with low-dose hydrogen peroxide in colorectal cancer cells in vitro(Basel : MDPI AG, 2020) Freund, Eric; Liedtke, Kim-Rouven; Miebach, Lea; Wende, Kristian; Heidecke, Amanda; Kaushik, Nagendra Kumar; Choi, Eun Ha; Partecke, Lars-Ivo; Bekeschus, SanderColorectal carcinoma is among the most common types of cancers. With this disease, diffuse scattering in the abdominal area (peritoneal carcinosis) often occurs before diagnosis, making surgical removal of the entire malignant tissue impossible due to a large number of tumor nodules. Previous treatment options include radiation and its combination with intraperitoneal heat-induced chemotherapy (HIPEC). Both options have strong side effects and are often poor in therapeutic efficacy. Tumor cells often grow and proliferate dysregulated, with enzymes of the protein kinase family often playing a crucial role. The present study investigated whether a combination of protein kinase inhibitors and low-dose induction of oxidative stress (using hydrogen peroxide, H2O2) has an additive cytotoxic effect on murine, colorectal tumor cells (CT26). Protein kinase inhibitors from a library of 80 substances were used to investigate colorectal cancer cells for their activity, morphology, and immunogenicity (immunogenic cancer cell death, ICD) upon mono or combination. Toxic compounds identified in 2D cultures were confirmed in 3D cultures, and additive cytotoxicity was identified for the substances lavendustin A, GF109203X, and rapamycin. Toxicity was concomitant with cell cycle arrest, but except HMGB1, no increased expression of immunogenic markers was identified with the combination treatment. The results were validated for GF109203X and rapamycin but not lavendustin A in the 3D model of different colorectal (HT29, SW480) and pancreatic cancer cell lines (MiaPaca, Panc01). In conclusion, our in vitro data suggest that combining oxidative stress with chemotherapy would be conceivable to enhance antitumor efficacy in HIPEC. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemNon-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection(San Francisco, California, US : PLOS, 2021) Mohamed, Hager; Clemen, Ramona; Freund, Eric; Lackmann, Jan-Wilm; Wende, Kristian; Connors, Jennifer; Haddad, Elias K.; Dampier, Will; Wigdahl, Brian; Miller, Vandana; Bekeschus, Sander; Krebs, Fred C.; Kashanchi, FatahEffective control of infection by human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), requires continuous and life-long use of anti-retroviral therapy (ART) by people living with HIV-1 (PLWH). In the absence of ART, HIV-1 reemergence from latently infected cells is ineffectively suppressed due to suboptimal innate and cytotoxic T lymphocyte responses. However, ART-free control of HIV-1 infection may be possible if the inherent immunological deficiencies can be reversed or restored. Herein we present a novel approach for modulating the immune response to HIV-1 that involves the use of non-thermal plasma (NTP), which is an ionized gas containing various reactive oxygen and nitrogen species (RONS). J-Lat cells were used as a model of latent HIV-1 infection to assess the effects of NTP application on viral latency and the expression of pro-phagocytic and pro-chemotactic damage-associated molecular patterns (DAMPs). Exposure of J-Lat cells to NTP resulted in stimulation of HIV-1 gene expression, indicating a role in latency reversal, a necessary first step in inducing adaptive immune responses to viral antigens. This was accompanied by the release of pro-inflammatory cytokines and chemokines including interleukin-1β (IL-1β) and interferon-γ (IFN-γ); the display of pro-phagocytic markers calreticulin (CRT), heat shock proteins (HSP) 70 and 90; and a correlated increase in macrophage phagocytosis of NTP-exposed J-Lat cells. In addition, modulation of surface molecules that promote or inhibit antigen presentation was also observed, along with an altered array of displayed peptides on MHC I, further suggesting methods by which NTP may modify recognition and targeting of cells in latent HIV-1 infection. These studies represent early progress toward an effective NTP-based ex vivo immunotherapy to resolve the dysfunctions of the immune system that enable HIV-1 persistence in PLWH.
- ItemShort- and long-term polystyrene nano- and microplastic exposure promotes oxidative stress and divergently affects skin cell architecture and Wnt/beta-catenin signaling(London : BioMed Central, 2023) Schmidt, Anke; da Silva Brito, Walison Augusto; Singer, Debora; Mühl, Melissa; Berner, Julia; Saadati, Fariba; Wolff, Christina; Miebach, Lea; Wende, Kristian; Bekeschus, SanderNano- and microplastic particles (NMP) are strong environmental contaminants affecting marine ecosystems and human health. The negligible use of biodegradable plastics and the lack of knowledge about plastic uptake, accumulation, and functional consequences led us to investigate the short- and long-term effects in freshly isolated skin cells from mice. Using fluorescent NMP of several sizes (200 nm to 6 µm), efficient cellular uptake was observed, causing, however, only minor acute toxicity as metabolic activity and apoptosis data suggested, albeit changes in intracellular reactive species and thiol levels were observed. The internalized NMP induced an altered expression of various targets of the nuclear factor-2-related transcription factor 2 pathway and were accompanied by changed antioxidant and oxidative stress signaling responses, as suggested by altered heme oxygenase 1 and glutathione peroxide 2 levels. A highly increased beta-catenin expression under acute but not chronic NMP exposure was concomitant with a strong translocation from membrane to the nucleus and subsequent transcription activation of Wnt signaling target genes after both single-dose and chronic long-term NMP exposure. Moreover, fibroblast-to-myofibroblast transdifferentiation accompanied by an increase of α smooth muscle actin and collagen expression was observed. Together with several NMP-induced changes in junctional and adherence protein expression, our study for the first time elucidates the acute and chronic effects of NMP of different sizes in primary skin cells' signaling and functional biology, contributing to a better understanding of nano- and microplastic to health risks in higher vertebrates.
- ItemPancreatic Cancer Cells Undergo Immunogenic Cell Death upon Exposure to Gas Plasma-Oxidized Ringers Lactate(Basel : MDPI, 2023) Miebach, Lea; Mohamed, Hager; Wende, Kristian; Miller, Vandana; Bekeschus, SanderSurvival rates among patients with pancreatic cancer, the most lethal gastrointestinal cancer, have not improved compared to other malignancies. Early tumor dissemination and a supportive, cancer-promoting tumor microenvironment (TME) limit therapeutic options and consequently impede tumor remission, outlining an acute need for effective treatments. Gas plasma-oxidized liquid treatment showed promising preclinical results in other gastrointestinal and gynecological tumors by targeting the tumor redox state. Here, carrier solutions are enriched with reactive oxygen (ROS) and nitrogen (RNS) species that can cause oxidative distress in tumor cells, leading to a broad range of anti-tumor effects. Unfortunately, clinical relevance is often limited, as many studies have forgone the use of medical-grade solutions. This study investigated the efficacy of gas plasma-oxidized Ringer’s lactate (oxRilac), a physiological solution often used in clinical practice, on two pancreatic cancer cell lines to induce tumor toxicity and provoke immunogenicity. Tumor toxicity of the oxRilac solutions was further confirmed in three-dimensional tumor spheroids monitored over 72 h and in ovo using stereomicroscope imaging of excised GFP-expressing tumors. We demonstrated that cell death signaling was induced in a dose-dependent fashion in both cell lines and was paralleled by the increased surface expression of key markers of immunogenic cell death (ICD). Nuclear magnetic resonance (NMR) spectroscopy analysis suggested putative reaction pathways that may cause the non-ROS related effects. In summary, our study suggests gas plasma-deposited ROS in clinically relevant liquids as an additive option for treating pancreatic cancers via immune-stimulating and cytotoxic effects.
- 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.
- 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.
- 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.
- 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
- ItemGas Plasma Exposure of Glioblastoma Is Cytotoxic and Immunomodulatory in Patient-Derived GBM Tissue(Basel : MDPI, 2022) Bekeschus, Sander; Ispirjan, Mikael; Freund, Eric; Kinnen, Frederik; Moritz, Juliane; Saadati, Fariba; Eckroth, Jacqueline; Singer, Debora; Stope, Matthias B.; Wende, Kristian; Ritter, Christoph A.; Schroeder, Henry W. S.; Marx, SaschaGlioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor. Therapeutic options for glioblastoma are maximal surgical resection, chemotherapy, and radiotherapy. Therapy resistance and tumor recurrence demand, however, new strategies. Several experimental studies have suggested gas plasma technology, a partially ionized gas that generates a potent mixture of reactive oxygen species (ROS), as a future complement to the existing treatment arsenal. However, aspects such as immunomodulation, inflammatory consequences, and feasibility studies using GBM tissue have not been addressed so far. In vitro, gas plasma generated ROS that oxidized cells and led to a treatment time-dependent metabolic activity decline and G2 cell cycle arrest. In addition, peripheral blood-derived monocytes were co-cultured with glioblastoma cells, and immunomodulatory surface expression markers and cytokine release were screened. Gas plasma treatment of either cell type, for instance, decreased the expression of the M2-macrophage marker CD163 and the tolerogenic molecule SIGLEC1 (CD169). In patient-derived GBM tissue samples exposed to the plasma jet kINPen ex vivo, apoptosis was significantly increased. Quantitative chemokine/cytokine release screening revealed gas plasma exposure to significantly decrease 5 out of 11 tested chemokines and cytokines, namely IL-6, TGF-β, sTREM-2, b-NGF, and TNF-α involved in GBM apoptosis and immunomodulation. In summary, the immuno-modulatory and proapoptotic action shown in this study might be an important step forward to first clinical observational studies on the future discovery of gas plasma technology’s potential in neurosurgery and neuro-oncology especially in putative adjuvant or combinatory GBM treatment settings.