Iron from the gut: the role of divalent metal transporter 1.

Mammalian cells contain thousands of metalloproteins and evolved systems to correctly incorporate metal cofactors into their designated sites. Among the transient metals in living cells, iron is the most abundant element that present as an iron sulfur cluster, mono- and dinuclear iron centers or heme for catalytic reactions. Iron homeostasis is tightly regulated by intestinal iron absorption in mammals owing to the lack of an iron excretive transport system, apart from superficial epithelial cell detachment and urinary outflow reabsorptive impairment. In mammals, the central site for iron absorption is in the duodenum, where the divalent metal transporter 1 is essential for iron uptake. The most notable manifestation of mutated divalent metal transporter 1 presents as iron deficiency anemia in humans. In contrast, the mutation of ferroportin, which exports iron, causes iron overload by either gain or loss of function. Furthermore, hepcidin secretion from the liver suppresses iron efflux by internalizing and degrading ferroportin; thus, the hepcidin/ferroportin axis is extensively investigated for its potential as a therapeutic target to treat iron overload. This review focuses on the divalent metal transporter 1-mediated intestinal iron uptake and hepcidin/ferroportin axis that regulate systemic iron homeostasis.

Iron and Cancer: A Special Issue.

Iron is an essential element for all organisms, and iron-containing proteins play critical roles in cellular functions [...].

Low-temperature plasma as magic wand to differentiate between the good and the evil.

Plasma is the fourth physical state of matter, characterized by an ionized gaseous mixture, after solid, liquid, and gas phases, and contains a wide array of components such as ions, electrons, radicals, and ultraviolet ray. Whereas the sun and thunder are typical natural plasma, recent progress in the electronics enabled the generation of body-temperature plasma, designated as low-temperature plasma (LTP) or non-thermal plasma since the 1990s. LTP has attracted the attention of researchers for possible biological and medical applications. All the living species on earth utilize water as essential media for solvents and molecular transport. Thus, biological application of LTP naturally intervenes water whether LTP is exposed directly or indirectly, where plasma-activated lactate (PAL) is a standard, containing H2O2, NO2- and other identified molecules. Electron spin resonance and immunohistochemical studies demonstrated that LTP exposure is a handy method to load local oxidative stress. Cancer cells are characterized by persistent self-replication and high cytosolic catalytic Fe(II). Therefore, both direct exposure of LTP and PAL can provide higher damage to cancer cells in comparison to non-tumorous cells, which has been demonstrated in a variety of cancer types. The cell death mode is either apoptosis or ferroptosis, depending on the cancer-type. Thus, LTP and PAL are expected to work as an additional cancer therapy to the established guideline protocols, especially for use in somatic cavities or surgical margins.

Plasma activated Ringer's lactate solution.

Low-temperature plasma (LTP) has been widely used in life science. Plasma-activated solutions were defined as solutions irradiated with LTP, and water, medium, and Ringer's solutions have been irradiated with LTP to produce plasma-activated solutions. They contain chemical compounds produced by reactions among LTP, air, and solutions. Reactive oxygen and nitrogen species (RONS) are major components in plasma-activated solutions and recent studies revealed that plasma-activated organic compounds are produced in plasma-activated Ringer's lactate solution (PAL). Many in vitro and in vivo studies demonstrated that PAL exhibits anti-tumor effects on cancers, and biochemical analyses revealed intracellular molecular mechanisms of cancer cell death by PAL.

Non-thermal plasma elicits ferrous chloride-catalyzed DMPO-OH.

Non-thermal plasma (NTP) induces the generation of reactive oxygen species (ROS) and reactive nitrogen species, such as hydroxyl radicals (•OH), hydrogen peroxide (H2O2), singlet oxygen, superoxide, ozone, and nitric oxide, at near-physiological temperatures. These molecules promote blood coagulation, wound healing, disinfection, and selective cancer cell death. Based on these evidences, clinical trials of NTP have been conducted for treating chronic wounds and head and neck cancers. Although clinical applications have progressed, the stoichiometric quantification of NTP-induced ROS remains unclear in the liquid phase in the presence of FeCl2 or FeCl3 in combination with biocompatible reducing agents, which may modulate the final biological effects of NTP. In this study, we employed electron paramagnetic resonance spectroscopy to quantify ROS using spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and H2O2, using luminescent probe in the presence of FeCl2 or FeCl3. NTP-induced DMPO-OH levels were elevated 10-100 µM FeCl2 or 500 and 1000 µM FeCl3. NTP-induced DMPO-OH with 10 µM FeCl2 or FeCl3 was significantly scavenged by ascorbate, α-tocopherol, dithiothreitol, reduced glutathione, or oxidized glutathione, whereas dehydroascorbate was ineffective in 2 mM DMPO. NTP-induced H2O2 was significantly degraded by 100 µM FeCl2 and FeCl3 in an iron-dependent manner. Meanwhile, decomposition of H2O2 by catalase decayed DMPO-OH efficiently in the presence of iron, indicating iron causes DMPO-OH production and degradation simultaneously. These results suggest that NTP-induced DMPO-OH is generated by the H2O2-consuming, iron-dependent Fenton reaction and ferryl intermediates. The potential iron-mediated ROS production by NTP is also discussed to clarify the interaction between NTP-induced ROS and biomolecules.

The Role of Ferric Nitrilotriacetate in Renal Carcinogenesis and Cell Death: From Animal Models to Clinical Implications.

Iron is essential for cellular growth, and various ferroproteins and heme-containing proteins are involved in a myriad of cellular functions, such as DNA synthesis, oxygen transport, and catalytic reactions. As a consequence, iron deficiency causes pleiotropic effects, such as hypochromic microcytic anemia and growth disturbance, while iron overload is also deleterious by oxidative injury. To prevent the generation of iron-mediated reactive oxygen species (ROS), ferritin is synthesized to store excess iron in cells that are consistent with the clinical utility of the serum ferritin concentration to monitor the therapeutic effect of iron-chelation. Among the animal models exploring iron-induced oxidative stress, ferric nitrilotriacetate (Fe-NTA) was shown to initiate hepatic and renal lipid peroxidation and the development of renal cell carcinoma (RCC) after repeated intraperitoneal injections of Fe-NTA. Here, current understanding of Fe-NTA-induced oxidative stress mediated by glutathione-cycle-dependent iron reduction and the molecular mechanisms of renal carcinogenesis are summarized in combination with a summary of the relationship between the pathogenesis of human RCC and iron metabolism. In addition to iron-mediated carcinogenesis, the ferroptosis that is triggered by the iron-dependent accumulation of lipid peroxidation and is implicated in the carcinogenesis is discussed.

Tetrachloroaurate (III)-induced oxidation increases non-thermal plasma-induced oxidative stress.

Non-thermal plasma (NTP) devices have been explored for medical applications. NTP devices discharge electrons, positive ions, ultraviolet (UV), reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as the hydroxyl radical (•OH), singlet oxygen (1O2), superoxide (O2•-), hydrogen peroxide (H2O2), ozone, and nitric oxide, at near-physiological temperature. At preclinical stages or in human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral, and biofilm-related infections, wound healing, and cancer cell death. Here, we observed that ferric, vanadium, and gold(III) ions significantly elevated lipid peroxidation, which was measured by 2-thiobarbituric acid-reactive substances (TBARS) in combination with NTP exposure. Using 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) as a spin probe in electron paramagnetic resonance (EPR), we observed that tetrachloroaurate (III) yielded an M4PO-X spin adduct. Tetrachloroaurate-induced oxidation was attenuated efficiently by reduced (GSH) and oxidized glutathione (GSSG), while glycine (Gly), and L-glutamate (Glu), components of GSH, were ineffective. Furthermore, GSH and GSSG efficiently suppressed tetrachloroaurate-induced lipid peroxidation, while Gly and Glu were ineffective in suppressing TBARS elevation. These results indicate that tetrachloroaurate-induced oxidation is attenuated by GSH as well as GSSG. Further studies are warranted to elucidate the redox reactions between metal ions and biomolecules to advance the clinical application of NTP.

Asbestos-induced mesothelial injury and carcinogenesis: Involvement of iron and reactive oxygen species.

Asbestos fibers have been used as an industrial and construction material worldwide due to their high durability and low production cost. Commercial usage of asbestos is currently prohibited in Japan; however, the risk of asbestos-induced malignant mesothelioma (MM) remains. According to epidemiological data, the onset of MM is estimated to occur after a latent period of 30-40 years from initial exposure to asbestos fibers; thus, the continuous increase in MM is a concern. To explore the molecular mechanisms of MM using animal models, iron saccharate with iron chelator-induced sarcomatoid mesothelioma (SM) revealed hallmarks of homozygous deletion of Cdkn2a/2b by aCGH and microRNA-199/214 by expression microarray. Oral treatment of iron chelation by deferasirox decreased the rate of high-grade SM. Moreover, phlebotomy delayed MM development in crocidolite-induced MM in rats. In Divalent metal transporter 1 (Dmt1) transgenic mice, MM development was delayed because of low reactive oxygen species (ROS) production. These results indicate the importance of iron and ROS in mesothelial carcinogenesis. The aims of this review focus on the pathogenesis of elongated mineral particles (EMPs), including asbestos fibers and multiwalled carbon nanotubes (MWCNTs) that share similar rod-like shapes in addition to the molecular mechanisms of MM development.

Mice lacking DYRK2 exhibit congenital malformations with lung hypoplasia and altered Foxf1 expression gradient.

Congenital malformations cause life-threatening diseases in pediatrics, yet the molecular mechanism of organogenesis is poorly understood. Here we show that Dyrk2-deficient mice display congenital malformations in multiple organs. Transcriptome analysis reveals molecular pathology of Dyrk2-deficient mice, particularly with respect to Foxf1 reduction. Mutant pups exhibit sudden death soon after birth due to respiratory failure. Detailed analyses of primordial lungs at the early developmental stage demonstrate that Dyrk2 deficiency leads to altered airway branching and insufficient alveolar development. Furthermore, the Foxf1 expression gradient in mutant lung mesenchyme is disrupted, reducing Foxf1 target genes, which are necessary for proper airway and alveolar development. In ex vivo lung culture system, we rescue the expression of Foxf1 and its target genes in Dyrk2-deficient lung by restoring Shh signaling activity. Taken together, we demonstrate that Dyrk2 is essential for embryogenesis and its disruption results in congenital malformation.

Low temperature plasma irradiation products of sodium lactate solution that induce cell death on U251SP glioblastoma cells were identified.

Low-temperature plasma is being widely used in the various fields of life science, such as medicine and agriculture. Plasma-activated solutions have been proposed as potential cancer therapeutic reagents. We previously reported that plasma-activated Ringer's lactate solution exhibited selective cancer-killing effects, and that the plasma-treated L-sodium lactate in the solution was an anti-tumor factor; however, the components that are generated through the interactions between plasma and L-sodium lactate and the components responsible for the selective killing of cancer cells remain unidentified. In this study, we quantified several major chemical products, such as pyruvate, formate, and acetate, in plasma-activated L-sodium lactate solution by nuclear magnetic resonance analysis. We further identified novel chemical products, such as glyoxylate and 2,3-dimethyltartrate, in the solution by direct infusion-electrospray ionization with tandem mass spectrometry analysis. We found that 2,3-dimethyltartrate exhibited cytotoxic effects in glioblastoma cells, but not in normal astrocytes. These findings shed light on the identities of the components that are responsible for the selective cytotoxic effect of plasma-activated solutions on cancer cells, and provide useful data for the potential development of cancer treatments using plasma-activated L-sodium lactate solution.

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.

The therapeutic potential of curcumin in alleviating N-diethylnitrosamine and iron nitrilotriacetate induced renal cell tumours in mice via inhibition of oxidative stress: Implications for cancer chemoprevention.

This study was designed to reveal the protective effects of dietary supplementation of curcumin against renal cell tumours and oxidative stress induced by renal carcinogen iron nitrilotriacetate (Fe-NTA) in ddY male mice. The results showed that mice treated with a renal carcinogen, Fe-NTA, a 35% renal cell tumour incidence was noticed, whereas renal cell tumour occurrence was elevated to 80% in Fe-NTA promoted and N-diethylnitrosamine (DEN)-initiated mice as compared with saline- treated mice. No incidence of tumours has been observed in DEN-initiated non-promoted mice. Diet complemented with 0.5% and 1.0% curcumin fed prior to, during and after treatment with Fe-NTA in DEN-initiated animals, tumour incidence was reduced dose-dependently to about 45% and 30% respectively. Immunohistochemical studies also revealed the increased formation of 4-hydroxy-2-nonenal (HNE)-modified protein adducts and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in kidney tissue of mice treated with an intraperitoneal injection of Fe-NTA (6.0 mg Fe/kg body weight.). Furthermore, Fe-NTA treatment of mice also resulted in significant elevation of malondialdehyde (MDA), serum urea, and creatinine and decreases renal glutathione. However, the changes in most of these parameters were attenuated dose-dependently by prophylactic treatment of animals with 0.5% and 1% curcumin diet, this may be due to its antioxidative impact of curcumin. These results suggest that intake of curcumin is beneficial for the prevention of renal cell tumours and oxidative stress damage mediated by renal carcinogen, Fe-NTA.

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.

Carcinogenesis as Side Effects of Iron and Oxygen Utilization: From the Unveiled Truth toward Ultimate Bioengineering.

Evolution from the first life on earth to humans took ~3.8 billion years. During the time there have been countless struggles among the species. Mycobacterium tuberculosis was the last major uncontrollable species against the human public health worldwide. After the victory with antibiotics, cancer has become the leading cause of death since 1981 in Japan. Considering that life inevitably depends on ceaseless electron transfers through iron and oxygen, we believe that carcinogenesis is intrinsically unavoidable side effects of using iron and oxygen. Many animal models unequivocally revealed that excess iron is a risk for carcinogenesis. This is supported by a variety of human epidemiological data on cancer risk and prognosis. Cancer is basically a disease of the genome with persistently activated oncogenes and inactivated tumor suppressor genes through which iron addiction with ferroptosis-resistance is maintained. Engineering has made a great advance in the past 50 years. In particular, nanotechnology is distinct in that the size of the engineered molecules is similar to that of our biomolecules. While some nano-molecules are found carcinogenic, there are principles to avoid such carcinogenicity with a smart possibility to use nano-molecules to specifically kill cancer cells. Non-thermal plasma is another modality to fight against cancer.

Asbestos conceives Fe(II)-dependent mutagenic stromal milieu through ceaseless macrophage ferroptosis and ß-catenin induction in mesothelium.

Asbestos is still a social burden worldwide as a carcinogen causing malignant mesothelioma. Whereas recent studies suggest that local iron reduction is a preventive strategy against carcinogenesis, little is known regarding the cellular and molecular mechanisms surrounding excess iron. Here by differentially using high-risk and low-risk asbestos fibers (crocidolite and anthophyllite, respectively), we identified asbestos-induced mutagenic milieu for mesothelial cells. Rat and cell experiments revealed that phagocytosis of asbestos by macrophages results in their distinctive necrotic death; initially lysosome-depenent cell death and later ferroptosis, which increase intra- and extra-cellular catalytic Fe(II). DNA damage in mesothelial cells, as assessed by 8-hydroxy-2'-deoxyguanosine and γ-H2AX, increased after crocidolite exposure during regeneration accompanied by ß-catenin activation. Conversely, ß-catenin overexpression in mesothelial cells induced higher intracellular catalytic Fe(II) with increased G2/M cell-cycle fraction, when p16INK4A genomic loci localized more peripherally in the nucleus. Mesothelial cells after challenge of H2O2 under ß-catenin overexpression presented low p16INK4A expression with a high incidence of deletion in p16INK4A locus. Thus, crocidolite generated catalytic Fe(II)-rich mutagenic environment for mesothelial cells by necrotizing macrophages with lysosomal cell death and ferroptosis. These results suggest novel molecular strategies to prevent mesothelial carcinogenesis after asbestos exposure.

Overexpression of miR-199/214 is a distinctive feature of iron-induced and asbestos-induced sarcomatoid mesothelioma in rats.

Malignant mesothelioma (MM) is one of the most lethal tumors in humans. The onset of MM is linked to exposure to asbestos, which generates reactive oxygen species (ROS). ROS are believed to be derived from the frustrated phagocytosis and the iron in asbestos. To explore the pathogenesis of MM, peritoneal MM was induced in rats by the repeated intraperitoneal injection of iron saccharate and nitrilotriacetate. In the present study, we used microarray techniques to screen the microRNA (miR) expression profiles of these MM. We observed that the histological subtype impacted the hierarchical clustering of miR expression profiles and determined that miR-199/214 is a distinctive feature of iron saccharate-induced sarcomatoid mesothelioma (SM). Twist1, a transcriptional regulator of the epithelial-mesenchymal transition, has been shown to activate miR-199/214 transcription; thus, the expression level of Twist1 was examined in iron-induced and asbestos-induced mesotheliomas in rats. Twist1 was exclusively expressed in iron saccharate-induced SM but not in the epithelioid subtype. The Twist1-miR-199/214 axis is activated in iron saccharate-induced and asbestos-induced SM. The expression levels of miR-214 and Twist1 were correlated in an asbestos-induced MM cell line, suggesting that the Twist1-miR-199/214 axis is preserved. MeT5A, an immortalized human mesothelial cell line, was used for the functional analysis of miR. The overexpression of miR-199/214 promoted cellular proliferation, mobility and phosphorylation of Akt and ERK in MeT5A cells. These results indicate that miR-199/214 may affect the aggressive biological behavior of SM.

Mth1 deficiency provides longer survival upon intraperitoneal crocidolite injection in female mice.

Exposure to asbestos fiber is central to mesothelial carcinogenesis. Recent sequencing studies on human and rodent malignant mesothelioma (MM) revealed frequently mutated genes, including CDKN2A, BAP1 and NF2. Crocidolite directly or indirectly catalyses the generation of hydroxyl radicals, which appears to be the major driving force for mesothelial mutations. DNA base modification is an oxidative DNA damage mechanism, where 8-hydroxy-2'-deoxyguanosine (8-OHdG) is the most abundant modification both physiologically and pathologically. Multiple distinct mechanisms work together to decrease the genomic level of 8-OHdG through the enzymatic activities of Mutyh, Ogg1 and Mth1. Knockout of one or multiple enzymes is not lethal but increases the incidence of tumors. Here, we used single knockout (KO) mice to test whether the deficiency of these three genes affects the incidence and prognosis of asbestos-induced MM. Intraperitoneal injection of 3 mg crocidolite induced MM at a fraction of 14.8% (4/27) in Mth1 KO, 41.4% (12/29) in Mutyh KO and 24.0% (6/25) in Ogg1 KO mice, whereas 31.7% (20/63) induction was observed in C57BL/6 wild-type (Wt) mice. The lifespan of female Mth1 KO mice was longer than that of female Wt mice (p = 0.0468). Whole genome scanning of MM with array-based comparative genomic hybridization revealed rare genomic alterations compared to MM in rats and humans. These results indicate that neither Mutyh deficiency nor Ogg1 deficiency promotes crocidolite-induced MM in mice, but the sanitizing nucleotide pool with Mth1 is advantageous in crocidolite-induced mesothelial carcinogenesis.

Frequent homozygous deletion of Cdkn2a/2b in tremolite-induced malignant mesothelioma in rats.

The onset of malignant mesothelioma (MM) is linked to exposure to asbestos fibers. Asbestos fibers are classified as serpentine (chrysotile) or amphibole, which includes the crocidolite, amosite, anthophyllite, tremolite, and actinolite types. Although few studies have been undertaken, anthophyllite has been shown to be associated with mesothelioma, and tremolite, a contaminant in talc and chrysotile, is a risk factor for carcinogenicity. Here, after characterizing the length and width of these fibers by scanning electron microscopy, we explored the cytotoxicity induced by tremolite and anthophyllite in cells from an immortalized human mesothelial cell line (MeT5A), murine macrophages (RAW264.7), and in a rat model. Tremolite and short anthophyllite fibers were phagocytosed and localized to vacuoles, whereas the long anthophyllite fibers were caught on the pseudopod of the MeT5A and Raw 264.7 cells, according to transmission electron microscopy. The results from a 2-day time-lapse study revealed that tremolite was engulfed and damaged the MeT5A and RAW264.7 cells, but anthophyllite was not cytotoxic to these cells. Intraperitoneal injection of tremolite in rats induced diffuse serosal thickening, whereas anthophyllite formed focal fibrosis and granulomas on peritoneal serosal surfaces. Furthermore, the loss of Cdkn2a/2b, which are the most frequently lost foci in human MM, were observed in 8 cases of rat MM (homozygous deletion [5/8] and loss of heterozygosity [3/8]) by array-based comparative genomic hybridization techniques. These results indicate that tremolite initiates mesothelial injury and persistently frustrates phagocytes, causing subsequent peritoneal fibrosis and MM. The possible mechanisms of carcinogenicity based on fiber diameter/length are discussed.

Oxidative stress-dependent and -independent death of glioblastoma cells induced by non-thermal plasma-exposed solutions.

Non-thermal atmospheric pressure plasma has been widely used for preclinical studies in areas such as wound healing, blood coagulation, and cancer therapy. We previously developed plasma-activated medium (PAM) and plasma-activated Ringer's lactate solutions (PAL) for cancer treatments. Many in vitro and in vivo experiments demonstrated that both PAM and PAL exhibit anti-tumor effects in several types of cancer cells such as ovarian, gastric, and pancreatic cancer cells as well as glioblastoma cells. However, interestingly, PAM induces more intracellular reactive oxygen species in glioblastoma cells than PAL. To investigate the differences in intracellular molecular mechanisms of the effects of PAM and PAL in glioblastoma cells, we measured gene expression levels of antioxidant genes such as CAT, SOD2, and GPX1. Microarray and quantitative real-time PCR analyses revealed that PAM elevated stress-inducible genes that induce apoptosis such as GADD45α signaling molecules. PAL suppressed genes downstream of the survival and proliferation signaling network such as YAP/TEAD signaling molecules. These data reveal that PAM and PAL induce apoptosis in glioblastoma cells by different intracellular molecular mechanisms.

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.