Cold Atmospheric Plasma Apoptotic and Oxidative Effects on MCF7 and HCC1806 Human Breast Cancer Cells

Breast cancer (BC) is a malignant neoplasia with the highest incidence and mortality rates in women worldwide. Currently, therapies include surgery, radiotherapy, and chemotherapy, including targeted therapies in some cases. However, treatments are often associated with serious adverse effects. Looking for new options in BC treatment, we evaluated the therapeutic potential of cold atmospheric plasma (CAP) in two cell lines (MCF7 and HCC1806) with distinct histological features. Apoptosis seemed to be the most prevalent type of death, as corroborated by several biochemical features, including phosphatidylserine exposure, the disruption of mitochondrial membrane potential, an increase in BAX/BCL2 ratio and procaspase 3 loss. Moreover, the accumulation of cells in the G2/M phase of the cell cycle points to the loss of replication ability and decreased survival. Despite reported toxic concentrations of peroxides in culture media exposed to plasma, intracellular peroxide concentration was overall decreased accompanying a reduction in GSH levels shortly after plasma exposure in both cell lines. In HCC1806, elevated nitric oxide (NO) concentration accompanied by reduced superoxide levels suggests that these cells are capable of converting plasma-derived nitrites into NO that competes with superoxide dismutase (SOD) for superoxide to form peroxinitrite. The concomitant inhibition of the antioxidative activity of cells during CAP treatment, particularly the inhibition of cytochrome c oxidase with sodium azide, synergistically increased plasma toxicity. Thus, this in vitro research enlightens the therapeutic potential of CAP in the treatment of breast cancer, elucidating its possible mechanisms of action.     

Cold Atmospheric Plasma Jet Treatment Improves Human Keratinocyte Migration and Wound Closure Capacity without Causing Cellular Oxidative Stress

Cold Atmospheric Plasma (CAP) is an emerging technology with great potential for biomedical applications such as sterilizing equipment and antitumor strategies. CAP has also been shown to improve skin wound healing in vivo, but the biological mechanisms involved are not well known. Our study assessed a possible effect of a direct helium jet CAP treatment on keratinocytes, in both the immortalized N/TERT-1 human cell line and primary keratinocytes obtained from human skin samples. The cells were covered with 200 µL of phosphate buffered saline and exposed to the helium plasma jet for 10–120 s. In our experimental conditions, micromolar concentrations of hydrogen peroxide, nitrite and nitrate were produced. We showed that long-time CAP treatments (≥60 s) were cytotoxic, reduced keratinocyte migration, upregulated the expression of heat shock protein 27 (HSP27) and induced oxidative cell stress. In contrast, short-term CAP treatments (<60 s) were not cytotoxic, did not affect keratinocyte proliferation and differentiation, and did not induce any changes in mitochondria, but they did accelerate wound closure in vitro by improving keratinocyte migration. In conclusion, these results suggest that helium-based CAP treatments improve wound healing by stimulating keratinocyte migration. The study confirms that CAP could be a novel therapeutic method to treat recalcitrant wounds.     

Biocompatible Gas Plasma Treatment Affects Secretion Profiles but Not Osteogenic Differentiation in Patient-Derived Mesenchymal Stromal Cells

Cold physical plasma (CPP), a partially ionized gas that simultaneously generates reactive oxygen and nitrogen species, is suggested to provide advantages in regenerative medicine. Intraoperative CPP therapy targeting pathologies related to diminished bone quality could be promising in orthopedic surgery. Assessment of a clinically approved plasma jet regarding cellular effects on primary bone marrow mesenchymal stromal cells (hBM-MSCs) from relevant arthroplasty patient cohorts is needed to establish CPP-based therapeutic approaches for bone regeneration. Thus, the aim of this study was to derive biocompatible doses of CPP and subsequent evaluation of human primary hBM-MSCs’ osteogenic and immunomodulatory potential. Metabolic activity and cell proliferation were affected in a treatment-time-dependent manner. Morphometric high content imaging analyses revealed a decline in mitochondria and nuclei content and increased cytoskeletal compactness following CPP exposure. Employing a nontoxic exposure regime, investigation on osteogenic differentiation did not enhance osteogenic capacity of hBM-MSCs. Multiplex analysis of major hBM-MSC cytokines, chemokines and growth factors revealed an anti-inflammatory, promatrix-assembling and osteoclast-regulating secretion profile following CPP treatment and osteogenic stimulus. This study can be noted as the first in vitro study addressing the influence of CPP on hBM-MSCs from individual donors of an arthroplasty clientele.     

Differential Sensitivity of Melanoma Cells and Their Non-Cancerous Counterpart to Cold Atmospheric Plasma-Induced Reactive Oxygen and Nitrogen Species

Despite continuous progress in therapy, melanoma is one of the most aggressive and malignant human tumors, often relapsing and metastasizing to almost all organs. Cold atmospheric plasma (CAP) is a novel anticancer tool that utilizes abundant reactive oxygen and nitrogen species (RONS) being deposited on the target cells and tissues. CAP-induced differential effects between non-cancerous and cancer cells were comparatively examined. Melanoma and non-cancerous skin fibroblast cells (counterparts; both cell types were isolated from the same patient) were used for plasma–cell interactions. The production of intracellular RONS, such as nitric oxide (NO), hydroxyl radical (•OH), and hydrogen peroxide (H₂O₂), increased remarkably only in melanoma cancer cells. It was observed that cancer cells morphed from spread to round cell shapes after plasma exposure, suggesting that they were more affected than non-cancerous cells in the same plasma condition. Immediately after both cell types were treated with plasma, there were no differences in the amount of extracellular H₂O₂ production, while Hanks’ balanced salt solution-containing cancer cells had lower concentrations of H₂O₂ than that of non-cancerous cells at 1 h after treatment. The melanoma cells seemed to respond to CAP treatment with a greater rise in RONS and a higher consumption rate of H₂O₂ than homologous non-cancerous cells. These results suggest that differential sensitivities of non-cancerous skin and melanoma cells to CAP-induced RONS can enable the applicability of CAP in anticancer therapy.     

Open Questions in Cold Atmospheric Plasma Treatment in Head and Neck Cancer: A Systematic Review

Over the past decade, we witnessed a promising application of cold atmospheric plasma (CAP) in cancer therapy. The aim of this systematic review was to provide an exhaustive state of the art of CAP employed for the treatment of head and neck cancer (HNC), a tumor whose late diagnosis, local recurrence, distant metastases, and treatment failure are the main causes of patients’ death. Specifically, the characteristics and settings of the CAP devices and the in vitro and in vivo treatment protocols were summarized to meet the urgent need for standardization. Its molecular mechanisms of action, as well as the successes and pitfalls of current CAP applications in HNC, were discussed. Finally, the interesting emerging preclinical hypotheses that warrant further clinical investigation have risen. A total of 24 studies were included. Most studies used a plasma jet device (54.2%). Argon resulted as the mostly employed working gas (33.32%). Direct and indirect plasma application was reported in 87.5% and 20.8% of studies, respectively. In vitro investigations were 79.17%, most of them concerned with direct treatment (78.94%). Only eight (33.32%) in vivo studies were found; three were conducted in mice, and five on human beings. CAP showed pro-apoptotic effects more efficiently in tumor cells than in normal cells by altering redox balance in a way that oxidative distress leads to cell death. In preclinical studies, it exhibited efficacy and tolerability. Results from this systematic review pointed out the current limitations of translational application of CAP in the urge of standardization of the current protocols while highlighting promising effects as supporting treatment in HNC.     

Effects of a Novel Cold Atmospheric Plasma Treatment of Titanium on the Proliferation and Adhesion Behavior of Fibroblasts

Cold plasma treatment increases the hydrophilicity of the surfaces of implants and may enhance their integration with the surrounding tissues. The implaPrep prototype device from Relyon Plasma generates cold atmospheric plasma via dielectric barrier discharge (DBD). In this study, titanium surfaces were treated with the implaPrep device for 20 s and assessed as a cell culture surface for fibroblasts. One day after seeding, significantly more cells were counted on the surfaces treated with cold plasma than on the untreated control titanium surface. Additionally, the viability assay revealed significantly higher viability on the treated surfaces. Morphological observation of the cells showed certain differences between the treated and untreated titanium surfaces. While conventional plasma devices require compressed gas, such as oxygen or argon, the implaPrep device uses atmospheric air as the gas source. It is, therefore, compact in size and simple to handle, and may provide a safe and convenient tool for treating the surfaces of dental implants, which may further improve the implantation outcome.     

The Effects of Cold Atmospheric Pressure Plasma on Germination Parameters,Enzyme Activities and Induction of DNA Damage in Barley

Climate change, environmental pollution and pathogen resistance to available chemical agents are part of the problems that the food industry has to face in order to ensure healthy food for people and livestock. One of the promising solutions to these problems is the use of cold atmospheric pressure plasma (CAPP). Plasma is suitable for efficient surface decontamination of seeds and food products, germination enhancement and obtaining higher yields in agricultural production. However, the plasma effects vary due to plasma source, treatment conditions and seed type. In our study, we tried to find the proper conditions for treatment of barley grains by diffuse coplanar surface barrier discharge, in which positive effects of CAPP, such as enhanced germination or decontamination effects, would be maximized and harmful effects, such as oxidation and genotoxic potential, minimized. Besides germination parameters, we evaluated DNA damage and activities of various germination and antioxidant enzymes in barley seedlings. Plasma exposure resulted in changes in germination parameters and enzyme activities. Longer exposures had also genotoxic effects. As such, our findings indicate that appropriate plasma exposure conditions need to be carefully optimized in order to preserve germination, oxidation balance and genome stability, should CAPP be used in agricultural practice.     

Acidified Nitrite Contributes to the Antitumor Effect of Cold Atmospheric Plasma on Melanoma Cells

Cold atmospheric plasma (CAP) is partially ionized gas near room temperature with previously reported antitumor effects. Despite extensive research and growing interest in this technology, active components and molecular mechanisms of CAP are not fully understood to date. We used Raman spectroscopy and colorimetric assays to determine elevated nitrite and nitrate levels after treatment with a MiniFlatPlaster CAP device. Previously, we demonstrated CAP-induced acidification. Cellular effects of nitrite and strong extracellular acidification were assessed using live-cell imaging of intracellular Ca²⁺ levels, cell viability analysis as well as quantification of p21 and DNA damage. We further characterized these observations by analyzing established molecular effects of CAP treatment. A synergistic effect of nitrite and acidification was found, leading to strong cytotoxicity in melanoma cells. Interestingly, protein nitration and membrane damage were absent after treatment with acidified nitrite, thereby challenging their contribution to CAP-induced cytotoxicity. Further, phosphorylation of ERK1/2 was increased after treatment with both acidified nitrite and indirect CAP. This study characterizes the impact of acidified nitrite on melanoma cells and supports the importance of RNS during CAP treatment. Further, it defines and evaluates important molecular mechanisms that are involved in the cancer cell response to CAP.     

Purinergic Signaling in Pancreas—From Physiology to Therapeutic Strategies in Pancreatic Cancer

The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y₂ and P2Y₁₂ receptors, as well as A₂ receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.     

Melatonin Induces Apoptosis and Modulates Cyclin Expression and MAPK Phosphorylation in Pancreatic Stellate Cells Subjected to Hypoxia

In certain diseases of the pancreas, pancreatic stellate cells form an important part of fibrosis and are critical for the development of cancer cells. A hypoxic condition develops within the tumor, to which pancreatic stellate cells adapt and are able to proliferate. The consequence is the growth of the tumor. Melatonin, the product of the pineal gland, is gaining attention as an agent with therapeutic potential against pancreatic cancers. Its actions on tumor cells lead, in general, to a reduction in cell viability and proliferation. However, its effects on pancreatic stellate cells subjected to hypoxia are less known. In this study, we evaluated the actions of pharmacological concentrations of melatonin (1 mM–1 µM) on pancreatic stellate cells subjected to hypoxia. The results show that melatonin induced a decrease in cell viability at the highest concentrations tested. Similarly, the incorporation of BrdU into DNA was diminished by melatonin. The expression of cyclins A and D also was decreased in the presence of melatonin. Upon treatment of cells with melatonin, increases in the expression of major markers of ER stress, namely BIP, phospho-eIF2α and ATF-4, were detected. Modulation of apoptosis was noticed as an increase in caspase-3 activation. In addition, changes in the phosphorylated state of p44/42, p38 and JNK MAPKs were detected in cells treated with melatonin. A slight decrease in the content of α-smooth muscle actin was detected in cells treated with melatonin. Finally, treatment of cells with melatonin decreased the expression of matrix metalloproteinases 2, 3, 9 and 13. Our observations suggest that melatonin, at pharmacological concentrations, diminishes the proliferation of pancreatic stellate cells subjected to hypoxia through modulation of cell cycle, apoptosis and the activation of crucial MAPKs. Cellular responses might involve certain ER stress regulator proteins. In view of the results, melatonin could be taken into consideration as a potential therapeutic agent for pancreatic fibrosis.     

Digitoxin Affects Metabolism,ROS Production and Proliferation in Pancreatic Cancer Cells Differently Depending on the Cell Phenotype

Digitoxin has repeatedly shown to have negative effects on cancer cell viability; however, the actual mechanism is still unknown. In this study, we investigated the effects of digitoxin (1–100 nM) in four pancreatic cancer cell lines, BxPC-3, CFPAC-1, Panc-1, and AsPC-1. The cell lines differ in their KRAS/BRAF mutational status and primary tumor or metastasis origin. We could detect differences in the basal rates of cell proliferation, glycolysis, and ROS production, giving the cell lines different phenotypes. Digitoxin treatment induced apoptosis in all four cell lines, but to different degrees. Cells derived from primary tumors (Panc-1 and BxPC-3) were highly proliferating with a high proportion of cells in the S/G2 phase, and were more sensitive to digitoxin treatment than the cell lines derived from metastases (CFPAC-1 and AsPC-1), with a high proportion of cells in G0/G1. In addition, the effects of digitoxin on the rate of glycolysis, ROS production, and proliferation were dependent on the basal metabolism and origin of the cells. The KRAS downstream signaling pathways were not altered by digitoxin treatment, thus the effects exerted by digitoxin were probably disconnected from these signaling pathways. We conclude that digitoxin is a promising treatment in highly proliferating pancreatic tumors.     

Reactive Oxygen Species Released from Hypoxic Hepatocytes Regulates MMP-2 Expression in Hepatic Stellate Cells

Hypoxia is a common environmental stress factor and is associated with fibrogenesis. Matrix metalloproteinase-2 (MMP-2), produced by hepatic stellate cells (HSCs), plays an important role in liver fibrogenesis. However, inconsistent results have been reported on the impact of hypoxia on MMP-2 expression and activity in HSCs. We speculated that cell-cell interaction is involved in the regulation of MMP-2 expression and activity at low oxygen level in vivo. Therefore, in this report we investigated the mechanism by which hypoxic hepatocytes regulates MMP-2 expression in HSCs. Our results showed that the conditioned medium from hypoxia-treated rat hepatocytes strongly induced the expression of MMP-2 mRNA and protein in rat HSC-T6 cells. Reduced glutathione neutralized ROS released from hypoxic hepatocytes, leading to reduced MMP-2 expression in HSC-T6 cells. In addition, phospho-IκB-α protein level was increased in HSC-T6 cells treated with hypoxia conditioned medium, and NF-κB signaling inhibitor inhibited MMP-2 expression in HSC-T6 cells. Taken together, our data suggest that ROS is an important factor released by hypoxic hepatocytes to regulate MMP-2 expression in HSCs, and NF-κB signaling is crucially involved in ROS-induced MMP-2 expression in HSCs. Our findings suggest that strategies aimed at antagonizing the generation of ROS in hypoxic hepatocytes and inhibiting NF-κB signaling in HSCs may represent novel therapeutic options for liver fibrosis.     

Effect of Argon-Based Atmospheric Pressure Plasma Treatment on Hard Tissue Formation on Titanium Surface

In this paper, we suggest that the atmospheric pressure plasma treatment of pure titanium metal may be useful for improving the ability of rat bone marrow cells (RBMCs) to induce hard tissue differentiation. Previous studies have reported that the use of argon gas induces a higher degree of hard tissue formation. Therefore, this study compares the effects of plasma treatment with argon gas on the initial adhesion ability and hard tissue differentiation-inducing ability of RBMCs. A commercially available titanium metal plate was used as the experimental material. A plate polished using water-resistant abrasive paper #1500 was used as the control, and a plate irradiated with argon mixed with atmospheric pressure plasma was used as the experimental plate. No structural change was observed on the surface of the titanium metal plate in the scanning electron microscopy results, and no change in the surface roughness was observed via scanning probe microscopy. X-ray photoelectron spectroscopy showed a decrease in the carbon peak and the formation of hydroxide in the experimental group. In the distilled water drop test, a significant decrease in the contact angle was observed for the experimental group, and the results indicated superhydrophilicity. Furthermore, the bovine serum albumin adsorption, initial adhesion of RBMCs, alkaline phosphatase activity, calcium deposition, and genetic marker expression of rat bone marrow cells were higher in the experimental group than those in the control group at all time points. Rat distal femur model are used as in vivo model. Additionally, microcomputed tomography analysis showed significantly higher results for the experimental group, indicating a large amount of the formed hard tissue. Histopathological evaluation also confirmed the presence of a prominent newly formed bone seen in the images of the experimental group. These results indicate that the atmospheric pressure plasma treatment with argon gas imparts superhydrophilicity, without changing the properties of the pure titanium plate surface. It was also clarified that it affects the initial adhesion of bone marrow cells and the induction of hard tissue differentiation.     

Cold Atmospheric Plasma Changes the Amino Acid Composition of Solutions and Influences the Anti-Tumor Effect on Melanoma Cells

Cold Atmospheric Plasma (CAP) is an ionized gas near room temperature. Its anti-tumor effect can be transmitted either by direct treatment or mediated by a plasma-treated solution (PTS), such as treated standard cell culture medium, which contains different amino acids, inorganic salts, vitamins and other substances. Despite extensive research, the active components in PTS and its molecular or cellular mechanisms are not yet fully understood. The purpose of this study was the measurement of the reactive species in PTS and their effect on tumor cells using different plasma modes and treatment durations. The PTS analysis yielded mode- and dose-dependent differences in the production of reactive oxygen and nitrogen species (RONS), and in the decomposition and modification of the amino acids Tyrosine (Tyr) and Tryptophan (Trp). The Trp metabolites Formylkynurenine (FKyn) and Kynurenine (Kyn) were produced in PTS with the 4 kHz (oxygen) mode, inducing apoptosis in Mel Im melanoma cells. Nitrated derivatives of Trp and Tyr were formed in the 8 kHz (nitrogen) mode, elevating the p16 mRNA expression and senescence-associated ß-Galactosidase staining. In conclusion, the plasma mode has a strong impact on the composition of the active components in PTS and affects its anti-tumor mechanism. These findings are of decisive importance for the development of plasma devices and the effectiveness of tumor treatment.     

Celastrol Prevents Oxidative Stress Effects on FSHR,PAPP, and CYP19A1 Gene Expression in Cultured Human Granulosa-Lutein Cells

Regulation of oxidative stress (OS) is important to prevent damage to female reproductive physiology. While normal OS levels may have a regulatory role, high OS levels may negatively affect vital processes such as folliculogenesis or embryogenesis. The aim of this work was to study OS induced by glucose, a reactive oxygen species generator, or peroxynitrite, a reactive nitrogen species generator, in cultured human granulosa-lutein (hGL) cells from oocyte donors, analyzing expression of genes involved in oocyte maturation (FSHR, PAPP, and CYP19A1) and OS damage response (ALDH3A2). We also evaluated the effect of celastrol as an antioxidant. Our results showed that although both glucose and peroxynitrite produce OS increments in hGL cells, only peroxynitrite treatment increases ALDH3A2 and PAPP gene expression levels and decreases FSHR gene expression levels. Celastrol pre-treatment prevents this effect of peroxynitrite. Interestingly, when celastrol alone was added, we observed a reduction of the expression of all genes studied, which was independent of both OS inductors. In conclusion, regulation of OS imbalance by antioxidant substances such as celastrol may prevent negative effects of OS in female fertility. In addition to the antioxidant activity, celastrol may well have an independent role on regulation of gene expression in hGL cells.     

Argon Plasma Exposure Augments Costimulatory Ligands and Cytokine Release in Human Monocyte-Derived Dendritic Cells

Cold 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.     

Cold Atmospheric Pressure Plasma-Activated Medium Induces Selective Cell Death in Human Hepatocellular Carcinoma Cells Independently of Singlet Oxygen,Hydrogen Peroxide,Nitric Oxide and Nitrite/Nitrate

Cold atmospheric pressure plasma (CAP) and plasma-activated medium (PAM) induce cell death in diverse cancer cells and may function as powerful anti-cancer agents. The main components responsible for the selective anti-cancer effects of CAP and PAM remain elusive. CAP or PAM induces selective cell death in hepatocellular carcinoma cell lines Hep3B and Huh7 containing populations with cancer stem cell markers. Here, we investigated the major component(s) of CAP and PAM for mediating the selective anti-proliferative effect on Hep3B and Huh7 cells. The anti-proliferative effect of CAP was mediated through the medium; however, the reactive oxygen species scavenger N-acetyl cysteine did not suppress PAM-induced cell death. Neither high concentrations of nitrite or nitrite/nitrate nor a low concentration of H₂O₂ present in the PAM containing sodium pyruvate affected the viability of Hep3B and Huh7 cells. Inhibitors of singlet oxygen, superoxide anions, and nitric oxide retained the capacity of PAM to induce anti-cancer effects. The anti-cancer effect was largely blocked in the PAM prepared by placing an aluminum metal mesh, but not a dielectric PVC mesh, between the plasma source and the medium. Hence, singlet oxygen, hydrogen peroxide, nitric oxide, and nitrite/nitrate are not the main factors responsible for PAM-mediated selective death in Hep3B and Huh7 cells. Other factors, such as charged particles including various ions in CAP and PAM, may induce selective anti-cancer effects in certain cancer cells.