Ferroptosis induced by plasma-activated Ringer's lactate solution prevents oral cancer progression.

OBJECTIVE: This study aimed to investigate the effect of plasma-activated Ringer's lactate solution (PAL) on oral squamous cell carcinoma (OSCC) cells and carcinogenic processes with a particular focus on iron and collagenous matrix formation. MATERIALS AND METHODS: We used three OSCC cell lines, one keratinocyte cell line, and two fibroblast lines, and cell viability assays, immunoblotting, flow cytometry, and transmission electron microscopy were performed to evaluate the effect and type of cell death. The effect of PAL treatment on lysyl oxidase (LOX) expression was investigated in vitro and in vivo. Tamoxifen-inducible Mob1a/b double-knockout mice were used for the in vivo experiment. RESULTS: PAL killed OSCC cells more effectively than the control nontumorous cells and suppressed cell migration and invasion. Ferroptosis occurred and the protein level of LOX was downregulated in cancer cells in vitro and in vivo. Additionally, PAL improved the survival rate of mice and suppressed collagenous matrix formation. CONCLUSIONS: We demonstrated that PAL specifically kills OSCC cells and that ferroptosis occurs in vitro and in vivo. Furthermore, PAL can prevent carcinogenesis and improve the survival rate of oral cancer, especially tongue cancer, by changing collagenous matrix formation via LOX suppression.

Study of optical emission spectroscopy using modified Boltzmann plot in dual-frequency synchronized pulsed capacitively coupled discharges with DC bias at low-pressure in Ar/O2/C4F8 plasma etching process.

We consider the corona model and local thermal equilibrium approximations of a real plasma to measure the electron temperature (Te) and density (ne), respectively, using the optical emission spectroscopy (OES) method in dual-frequency pulsed capacitively coupled plasmas (CCPs) in a reactive mixture of Ar/O2/C4F8 at a low operating pressure. The operation conditions such as DC continuous and synchronized were used for the study and plasma characterization for the intended plasma application such as high aspect ratio etching (HARE). We show that the present plasma conditions are dominated by a corona balance rather than the supremacy of multi-step excitation. This fact has enabled us to utilize the modified Boltzmann plot technique to evaluate the Te values. In the second method, we simultaneously used the Boltzmann and Saha equations to determine the ne value using the line intensity ratio and the value of Te. Time-resolved measurements of Te and ne were performed for completeness, and the insight of the pulsed discharge was investigated. Time evolution of ne and Te using the OES method revealed a similar trend in the change of plasma parameters, indicating electron impact ionization during the pulse on phase. It was seen that ne in the afterglow speedily decreased within a short time of ∼5 µs. Analysis suggests the formation of afterglow plasmas, which are composed of positive and negative ions with very low electron density. The results revealed that the DC-synchronized operation could be useful for plasma application such as HARE due to different plasma characteristics. It also suggests the production of ion-ion plasmas by the effective utilization of negative ions in the afterglow phase. The corona balance condition was validated in our experiments, and the results were compared with the existing literature.

Non-thermal plasma-induced DMPO-OH yields hydrogen peroxide.

Recent developments in electronics have enabled the medical applications of non-thermal plasma (NTP), which elicits reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydroxyl radical (●OH), hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide (O2●-), ozone, and nitric oxide at near-physiological temperatures. In preclinical studies or human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral and biofilm-related infections, wound healing, and cancer cell death. To elucidate the solution-phase biological effects of NTP in the presence of biocompatible reducing agents, we employed electron paramagnetic resonance (EPR) spectroscopy to quantify ●OH using a spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); 1O2 using a fluorescent probe; and O2●- and H2O2 using luminescent probes in the presence of thiols or tempol. NTP-induced ●OH was significantly scavenged by dithiothreitol (DTT), reduced glutathione (GSH), and oxidized glutathione (GSSG) in 2 or 5 mM DMPO. NTP-induced O2●- was significantly scavenged by 10 µM DTT and GSH, while 1O2 was not efficiently scavenged by these compounds. GSSG degraded H2O2 more effectively than GSH and DTT, suggesting that the disulfide bonds reacted with H2O2. In the presence of 1-50 mM DMPO, NTP-induced H2O2 quantities were unchanged. The inhibitory effect of tempol concentration (50 and 100 µM) on H2O2 production was observed in 1 and 10 mM DMPO, whereas it became ineffective in 50 mM DMPO. Furthermore, DMPO-OH did not interact with tempol. These results suggest that DMPO and tempol react competitively with O2●-. Further studies are warranted to elucidate the interaction between NTP-induced ROS and biomolecules.

L-Dehydroascorbate efficiently degrades non-thermal plasma-induced hydrogen peroxide.

Non-thermal plasma (NTP) devices generate reactive oxygen species (ROS) and reactive nitrogen species, such as singlet oxygen (1O2), superoxide (O2-), hydroxyl radical (●OH), hydrogen peroxide (H2O2), ozone, and nitric oxide at near-physiological temperature. In preclinical studies, NTP promotes blood coagulation, wound healing with disinfection, and selective killing of cancer cells. Although these biological effects of NTP have been widely explored, the stoichiometric quantitation of ROS in the liquid phase has not been performed in the presence of biocompatible reducing agents, which may modify the final biological effects of NTP. Here, we utilized electron paramagnetic resonance spectroscopy to quantitate ●OH, using a spin-trapping probe 5,5-dimethyl-1-pyrroline-N-oxide; 1O2, using a fluorescent probe; and O2- and H2O2, using luminescent probes, after NTP exposure in the presence of antioxidants. l-ascorbate (Asc) at 50 µM concentration (physiological concentration in serum) significantly scavenged ●OH, whereas (-)-epigallocatechin gallate (EGCG) and α-tocopherol were also effective at performing scavenging activities at 250 µM concentrations. Asc significantly scavenged O2- and H2O2 at 100 µM. l-Dehydroascorbate (DHA), an oxidized form of Asc, degraded H2O2, whereas it did not quench ●OH or O2-, which are sources of H2O2. Furthermore, EGCG efficiently scavenged NTP-induced 1O2, O2-, and H2O2 in Chelex-treated water. These results indicate that the redox cycling of Asc/DHA and metabolites of DHA are important to be considered when applying NTP to cells and tissues. Additionally, ROS-reducing compounds, such as EGCG, affect the outcome. Further studies are warranted to elucidate the interaction between ROS and biomolecules to promote the medical applications of NTP.

Non-thermal plasma-activated lactate solution kills U251SP glioblastoma cells in an innate reductive manner with altered metabolism.

Ringer's lactate solution irradiated by non-thermal plasma, comprised of radicals, electrons, and ions, is defined as plasma-activated lactate (PAL). PAL exhibited antitumor effects in glioblastoma U251SP cells, which we termed PAL-specific regulated cell death. In contrast to the oxidative stress condition typical of cells incubated in plasma-activated medium (PAM), U251SP cells treated with Ringer's lactate solution or PAL exhibited changes in intracellular metabolites that were reductive in the redox state, as measured by the ratio of oxidative/reductive glutathione concentrations. In the metabolomic profiles of PAL-treated cells, the generation of acetyl-CoA increased for lipid metabolism from alanine and asparagine. PAL thus induces regulated death of U251SP glioblastoma cells in more innate microenvironments than PAM.

l-Dehydroascorbic acid recycled by thiols efficiently scavenges non-thermal plasma-induced hydroxyl radicals.

Recent development in electronics has enabled the use of non-thermal plasma (NTP) to strictly direct oxidative stress in a defined location at near-physiological temperature. In preclinical studies or human clinical trials, NTP promotes blood coagulation, wound healing with disinfection, and selective killing of cancer cells. Although these biological effects of NTP have been widely explored, the stoichiometric quantitation of free radicals in liquid phase has not been performed in the presence of biocompatible reducing agents, which may modify the final biological effects of NTP. Here we quantitated hydroxyl radicals, a major reactive oxygen species generated after NTP exposure, by electron paramagnetic resonance (EPR) spectroscopy using two distinct spin-trapping probes, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), in the presence of thiols or antioxidants. l-Ascorbic acid (AsA) at 25-50 µM concentrations (physiological concentration in the serum) significantly scavenged these hydroxyl radicals, whereas dithiothreitol (DTT), reduced glutathione (GSH), and N-acetyl-cysteine (NAC) as thiols were required in millimolar concentrations to perform scavenging activities. l-Dehydroascorbic acid (DHA), an oxidized form of AsA, necessitated the presence of 25-50 µM DTT or sub-millimolar concentrations of GSH and NAC for the scavenging of hydroxyl radicals and failed to scavenge hydroxyl radicals by itself. These results suggest that the redox cycling of AsA/DHA via thiols and cellular AsA metabolism are important processes to be considered while applying NTP to cells and tissues. Further studies are warranted to elucidate the interaction between other reactive species generated by NTP and biomolecules to promote biological and medical applications of NTP.

Non-thermal plasma-activated medium modified metabolomic profiles in the glycolysis of U251SP glioblastoma.

Non-equilibrium atmospheric pressure plasma (NEAPP) is a mixture of radicals, electrons, anions, cations and light at near body temperature. Plasma-activated medium (PAM) is realized using NEAPP provided by engineered devices and irradiated to a cell culture medium for a period of 600 s. Glioblastoma cells U251SP cultivated in PAM previously indicated that antitumor effects induced PAM-specific apoptotic cell-death. Metabolomic profiles of a hundred intracellular metabolites were analyzed using capillary electrophoresis mass spectrometry. The metabolomic profiles of the PAM-treated U251SP cells were changed significantly with inhibition of the glycolysis pathway and with enhancement of the pentose phosphate pathway.

Non-thermal plasma specifically kills oral squamous cell carcinoma cells in a catalytic Fe(II)-dependent manner.

Oral cancer accounts for ~2% of all cancers worldwide, and therapeutic intervention is closely associated with quality of life. Here, we evaluated the effects of non-thermal plasma on oral squamous cell carcinoma cells with special reference to catalytic Fe(II). Non-thermal plasma exerted a specific killing effect on oral squamous cell carcinoma cells in comparison to fibroblasts. Furthermore, the effect was dependent on the amounts of catalytic Fe(II), present especially in lysosomes. After non-thermal plasma application, lipid peroxidation occurred and peroxides and mitochondrial superoxide were generated. Cancer cell death by non-thermal plasma was promoted dose-dependently by prior application of ferric ammonium citrate and prevented by desferrioxamine, suggesting the association of ferroptosis. Potential involvement of apoptosis was also observed with positive terminal deoxynucleaotidyl transferase-mediated dUTP nick end labeling and annexin V results. Non-thermal plasma exposure significantly suppressed the migratory, invasive and colony-forming abilities of squamous cell carcinoma cells. The oral cavity is easily observable; therefore, non-thermal plasma can be directly applied to the oral cavity to kill oral squamous cell carcinoma without damaging fibroblasts. In conclusion, non-thermal plasma treatment is a potential therapeutic option for oral cancer.

Molecular mechanisms of non-thermal plasma-induced effects in cancer cells.

Plasma is the fourth state of matter with higher energy than gas; non-thermal plasma (NTP) is currently available. As NTP is useful in sterilization, promoting wound healing and cancer treatments, the molecular mechanisms of plasma-induced effects in living cells and microorganisms are of significant interest in plasma medicine with medical-engineering collaboration. Molecular mechanisms of plasma-induced effects in cancer cells will be described in this minireview. Both direct and indirect methods to treat cancer cells with NTP have been developed. NTP interacts directly with not only cancer cells but also the liquids surrounding cancer cells and the immune cells that target them. Reactive oxygen and nitrogen species play key roles in NTP-induced effects; however, other mechanisms have been suggested. The complex interactions between NTP, cells and liquids have been extensively studied. In the future, details regarding NTP-induced effects on gene regulatory networks, signaling networks, and metabolic networks will be elucidated.

Effect of Plasma-Activated Lactated Ringer's Solution on Pancreatic Cancer Cells In Vitro and In Vivo.

BACKGROUND: The medical applications of nonequilibrium atmospheric pressure plasma in cancer therapy have attracted attention. We previously reported on the antitumor effect of plasma-activated medium. However, this approach requires plasma-activated liquids that are administrable to the human body. In this study, we produced plasma-activated lactated Ringer's solution (PAL) and evaluated its antitumor effect and mechanism. Furthermore, we evaluated the effect of the intraperitoneal administration of PAL using a peritoneal dissemination mouse tumor model. METHODS: The antitumor effect of PAL on pancreatic cancer cell lines was evaluated using proliferation and apoptosis assays. In addition, cellular reactive oxygen species (ROS) generation was examined. The role of ROS was assessed using a proliferation assay with N-acetyl cysteine (NAC). An adhesion assay was performed to evaluate the effect of PAL on cell adhesion. Finally, pancreatic cancer cells stably expressing luciferase (AsPC-1/CMV-Luc) were injected intraperitoneally into mice, followed by intraperitoneal injection of PAL. Peritoneal dissemination was monitored using in vivo bioluminescent imaging. RESULTS: The antitumor effect of PAL was shown in all cell lines in vitro. The TUNEL assay showed that PAL induced apoptosis. ROS uptake was observed in PAL-treated cells, and the antitumor effect was inhibited by NAC. Cell adhesion also was suppressed by PAL. The intraperitoneal administration of PAL suppressed the formation of peritoneal nodules in vivo. CONCLUSIONS: Our study demonstrated the antitumor effects of PAL in vitro and in vivo. Intraperitoneal administration of PAL may be a novel therapeutic option for peritoneal metastases.

Plasma-activated medium (PAM) kills human cancer-initiating cells.

Medical non-thermal plasma (NTP) treatments for various types of cancers have been reported. Cells with tumorigenic potential (cancer-initiating cells; CICs) are few in number in many types of tumors. CICs efficiently eliminate anti-cancer chemicals and exhibit high-level aldehyde dehydrogenase (ALDH) activity. We previously examined the effects of direct irradiation via NTP on cancer cells; even though we targeted CICs expressing high levels of ALDH, such treatment affected both non-CICs and CICs. Recent studies have shown that plasma-activated medium (PAM) (culture medium irradiated by NTP) selectively induces apoptotic death of cancer but not normal cells. Therefore, we explored the anti-cancer effects of PAM on CICs among endometrioid carcinoma and gastric cancer cells. PAM reduced the viability of cells expressing both low and high levels of ALDH. Combined PAM/cisplatin appeared to kill cancer cells more efficiently than did PAM or cisplatin alone. In a mouse tumor xenograft model, PAM exerted an anti-cancer effect on CICs. Thus, our results suggest that PAM effectively kills both non-CICs and CICs, as does NTP. Therefore, PAM may be a useful new anti-cancer therapy, targeting various cancer cells including CICs.

Intraperitoneal Administration of Plasma-Activated Medium: Proposal of a Novel Treatment Option for Peritoneal Metastasis From Gastric Cancer.

BACKGROUND: The administration of fluid irradiated with non-equilibrium atmospheric pressure plasma (NEAPP) has attracted much interest as a novel therapeutic method for cancer. The authors previously reported on the efficacy of plasma-activated medium (PAM) for treating cancer cell lines through the induction of apoptosis. In this study, the therapeutic effect of PAM was evaluated in vivo using a peritoneal metastasis mouse model. METHODS: Two gastric cancer cell lines were used in proliferation assays performed to optimize the production of PAM by changing the distance between the plasma source and the medium surface and by altering the volume of irradiated medium. Wound-healing and adhesion assays were conducted to determine the effect of PAM therapy on cell migration and adhesion capacity in vitro. Finally, a mouse model established by the intraperitoneal injection of enhanced green fluorescent protein-tagged gastric cancer cells was used to explore the efficacy of PAM administered intraperitoneally in inhibiting peritoneal metastasis formation. RESULTS: Shorter distances between the plasma source and the medium surface and smaller volumes of treated medium increased the anti-tumor effect of PAM. The PAM treatment attenuated gastric cancer cell migration and adhesion in vitro. The intraperitoneal administration of PAM decreased the formation of peritoneal metastatic nodules by 60% in the mouse model, and no adverse events were observed. CONCLUSIONS: Plasma-activated liquids may represent a novel therapeutic method for the treatment of peritoneal metastases in gastric cancer.

Intracellular-molecular changes in plasma-irradiated budding yeast cells studied using multiplex coherent anti-Stokes Raman scattering microscopy.

Interactions between non-equilibrium atmospheric-pressure plasma (NEAPP) and living cells were examined using multiplex coherent anti-Stokes Raman scattering (CARS) microscopy. Our multiplex CARS analyses revealed that NEAPP irradiation generates short-lived radicals that induce a decrease in the mitochondrial activity of budding yeast cells.

Medical applications of non-thermal atmospheric pressure plasma.

An innovative approach for producing reactive oxygen and nitrogen species is the use of non-thermal atmospheric pressure plasma. The technique has been applied in a wide variety of fields ranging from the micro-fabrication of electric devices to the treatment of disease. Although non-thermal atmospheric pressure plasmas have been shown to be clinically beneficial for wound healing, blood coagulation, and cancer treatment, the underlying molecular mechanisms are poorly understood. In this review, we describe the current progress in plasma medicine, with a particular emphasis on plasma-activated medium (PAM), which is a solution that is irradiated with a plasma and has broadened the applications of plasmas in medicine.

Future perspective of strategic non-thermal plasma therapy for cancer treatment.

The therapeutic effects of non-thermal plasma are expected in the medical fields, including hemostasis, vascularization, prevention of organ adhesion, and cell proliferation. Cancer is an internal enemy arising from normal tissue in the body. The prognosis of metastatic and recurrent cancers is still poor despite advances in medicine. To apply non-thermal plasma in cancer treatment is now on going. The mechanism of the proliferation-inhibitory effect of plasma is reactive nitrogen oxide species/reactive oxygen species production in cells. There are a number of problems to be overcome, such as existence of intrinsic reactive oxygen species/reactive nitrogen species scavengers and the shallow infiltration of plasma on tumor surface. The current reviews makes referral to the study results of plasma therapy clarified so far, the possibility of its application in the future.

Helium-based cold atmospheric plasma-induced reactive oxygen species-mediated apoptotic pathway attenuated by platinum nanoparticles.

Plasma is generated by ionizing gas molecules. Helium (He)-based cold atmospheric plasma (CAP) was generated using a high-voltage power supply with low-frequency excitation (60 Hz at 7 kV) and He flow at 2 l/min. Platinum nanoparticles (Pt-NPs) are potent antioxidants due to their unique ability to scavenge superoxides and peroxides. These features make them useful for the protection against oxidative stress-associated pathologies. Here, the effects of Pt-NPs on He-CAP-induced apoptosis and the underlying mechanism were examined in human lymphoma U937 cells. Apoptosis was measured after cells were exposed to He-CAP in the presence or absence of Pt-NPs. The effects of combined treatment were determined by observing the changes in intracellular reactive oxygen species (ROS) and both mitochondrial and Fas dependent pathway. The results indicate that Pt-NPs substantially scavenge He-CAP-induced superoxides and peroxides and inhibit all the pathways involved in apoptosis execution. This might be because of the SOD/catalase mimetic effects of Pt-NPs. These results showed that the Pt-NPs can induce He-CAP desensitization in human lymphoma U937 cells.

Nanopore formation process in artificial cell membrane induced by plasma-generated reactive oxygen species.

We investigated morphological change of an artificial lipid bilayer membrane induced by oxygen radicals which were generated by non-equilibrium atmospheric pressure plasma. Neutral oxygen species, O((3)Pj) and O2((1)Δg), were irradiated of a supported lipid bilayer existing under a buffer solution at various conditions of dose time and distances, at which the dose amounts of the oxygen species were calculated quantitatively. Observation using an atomic force microscope and a fluorescence microscope revealed that dose of the neutral oxygen species generated nanopores with the diameter of 10-50 nm in a phospholipid bilayer, and finally destructed the bilayer structure. We found that protrusions appeared on the lipid bilayer surface prior to the formation of nanopores, and we attributed the protrusions to the precursor of the nanopores. We propose a mechanism of the pore formation induced by lipid oxidation on the basis of previous experimental and theoretical studies.

Galectin expression in healing wounded skin treated with low-temperature plasma: Comparison with treatment by electronical coagulation.

Low-temperature plasma is useful for the care of wounded skin. It accelerates wound healing. However, the mechanism of this effect has not been fully elucidated yet. Galectin-1 is reported to accelerate wound healing via the Smad signaling pathway. In the present study to clarify whether or not galectins were expressed during the process of wound healing in the plasma-treated skin, we examined the effect of low-temperature plasma on galectin expression in the healing skin. We compared the effects of low-temperature plasma on the expression of galectin-1, -2, and -3 in the healing skin with those of electrocoagulation conducted with a high-frequency electrical coagulator. Immediately after the start of low-temperature plasma treatment following the incision made in the skin, a membrane-like structure was formed on the surface of the wound. Immunoelectron microscopy showed that these galectins were localized in the membrane-like structure of the plasma-treated skin. The expressions of these galectins were increased by the low-temperature plasma treatment, whereas they were inhibited by the electrocoagulation. These results suggest that galectins were involved in the wound healing of low-temperature plasma-treated skin. Galectins will thus be good markers for further examination of the effects of low-temperature plasma on the healing of wounded skin.

Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells.

Non-thermal plasma (NTP) is a recently developed technology that elicits a variety of biological effects. This includes cancer cell-specific cytotoxicity, which is mainly attributed to the regional generation of reactive oxygen species (ROS). We studied the effects of NTP on malignant mesothelioma (MM) and its modulation by l-ascorbate. l-ascorbate is a major water-soluble anti-oxidant in vivo, but its pro-oxidant activity in vitro has been well recognized. Thus, the effects of ascorbate on the efficacy of NTP is important to examine. NTP exposure dose-dependently killed MM cells, whereas MM cells tolerated 1 mM l-ascorbate. However, brief pre-treatment with a pharmacological dose (250-750 µM) of l-ascorbate immediately prior to NTP exposure significantly increased its cytotoxicity in a dose-dependent manner, which was inhibited by the iron chelator, deferoxamine. However, paradoxically, this potentiating effect of l-ascorbate was completely abolished by a prolonged 4 h pre-incubation with l-ascorbate (500 µM). MM cytotoxicity induced by NTP was associated with immediate oxidative stress evaluated by 2',7'-dichlorodihydrofluorecein diacetate, which was followed by an increase in the expression of the autophagosome marker, LC3B-II. In conclusion, MM can be a target for NTP treatment and l-ascorbate can increase or decrease its efficacy depending on the length of the pre-incubation period.

Low temperature plasma processing for cell growth inspired carbon thin films fabrication.

The recent bio-applications (i.e. bio-sensing, tissue engineering and cell proliferation etc.) are driving the fundamental research in carbon based materials with functional perspectives. High stability in carbon based coatings usually demands the high density deposition. However, the standard techniques, used for the large area and high throughput deposition of crystalline carbon films, often require very high temperature processing (typically >800 °C in inert atmosphere). Here, we present a low temperature (<150 °C) pulsed-DC plasma sputtering process, which enables sufficient ion flux to deposit dense unhydrogenated carbon thin films without any need of substrate-bias or post-deposition thermal treatments. It is found that the control over plasma power density and pulsed frequency governs the density and kinetic energy of carbon ions participating during the film growth. Subsequently, it controls the contents of sp(3) and sp(2) hybridizations via conversion of sp(2) to sp(3) hybridization by ion's energy relaxation. The role of plasma parameters on the chemical and surface properties are presented and correlated to the bio-activity. Bioactivity tests, carried out in mouse fibroblast L-929 and Sarcoma osteogenic (Saos-2) bone cell lines, demonstrate promising cell-proliferation in these films.