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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.
- 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.
- 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.
- 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.
- 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.
- 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.