Publications by authors named "Shinya Toyokuni"

261 Publications

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

Arch Biochem Biophys 2021 Jul 6;705:108901. Epub 2021 May 6.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan. Electronic address:

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 (HO), singlet oxygen (O), superoxide (O), 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); O using a fluorescent probe; and O and HO 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 O was significantly scavenged by 10 μM DTT and GSH, while O was not efficiently scavenged by these compounds. GSSG degraded HO more effectively than GSH and DTT, suggesting that the disulfide bonds reacted with HO. In the presence of 1-50 mM DMPO, NTP-induced HO quantities were unchanged. The inhibitory effect of tempol concentration (50 and 100 μM) on HO 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 O. Further studies are warranted to elucidate the interaction between NTP-induced ROS and biomolecules.
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http://dx.doi.org/10.1016/j.abb.2021.108901DOI Listing
July 2021

Lysosomal nitric oxide determines transition from autophagy to ferroptosis after exposure to plasma-activated Ringer's lactate.

Redox Biol 2021 Jul 23;43:101989. Epub 2021 Apr 23.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan; Center for Low Temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan; Sydney Medical School, The University of Sydney, Sydney, NSW, 2006, Australia. Electronic address:

Non-thermal plasma (NTP), an engineered technology to generate reactive species, induces ferroptosis and/or apoptosis specifically in various-type cancer cells. NTP-activated Ringer's lactate (PAL) is another modality for cancer therapy at preclinical stage. Here we found that PAL induces selective ferroptosis of malignant mesothelioma (MM) cells, where non-targeted metabolome screening identified upregulated citrulline-nitric oxide (NO) cycle as a PAL target. NO probe detected biphasic peaks transiently at PAL exposure with time-dependent increase, which was responsible for inducible NO synthase (iNOS) overexpression through NF-κB activation. NO and lipid peroxidation occupied lysosomes as a major compartment with increased TFEB expression. Not only ferrostatin-1 but inhibitors for NO and/or iNOS could suppress this ferroptosis. PAL-induced ferroptosis accompanied autophagic process in the early phase, as demonstrated by an increase in essential amino acids, LC3B-II, p62 and LAMP1, transforming into the later phase with boosted lipid peroxidation. Therefore, NO-mediated lysosomal impairment is central in PAL-induced ferroptosis.
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http://dx.doi.org/10.1016/j.redox.2021.101989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105670PMC
July 2021

Preclinical Verification of the Efficacy and Safety of Aqueous Plasma for Ovarian Cancer Therapy.

Cancers (Basel) 2021 Mar 7;13(5). Epub 2021 Mar 7.

Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. The major cause of EOC's lethality is that intraperitoneal recurrence occurs with high frequency due to occult metastasis. We had demonstrated that plasma-activated medium (PAM) exerts a metastasis-inhibitory effect on ovarian cancer in vitro and in vivo. Here we investigated how PAM inhibits intraperitoneal metastasis. We studied PAM's inhibition of micro-dissemination onto the omentum by performing in vivo imaging in combination with a sequential histological analysis. The results revealed that PAM induced macrophage infiltration into the disseminated lesion. The iNOS-positive signal was co-localized at the macrophages in the existing lesion, indicating that PAM might induce M1-type macrophages. This may be another mechanism of the antitumor effect through a PAM-evoked immune response. Intraperitoneal lavage with plasma-activated lactate Ringer's solution (PAL) significantly improved the overall survival rate in an ovarian cancer mouse model. Our results demonstrated the efficiency and practicality of aqueous plasma for clinical applications.
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http://dx.doi.org/10.3390/cancers13051141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962102PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation.

Cell Rep 2021 Feb;34(6):108734

Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Japan; Laboratory of Immunology and Microbiology, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Japan; PRESTO, JST, Kawaguchi, Japan. Electronic address:

Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.
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http://dx.doi.org/10.1016/j.celrep.2021.108734DOI Listing
February 2021

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

Arch Biochem Biophys 2021 03 20;700:108762. Epub 2021 Jan 20.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan. Electronic address:

Non-thermal plasma (NTP) devices generate reactive oxygen species (ROS) and reactive nitrogen species, such as singlet oxygen (O), superoxide (O), hydroxyl radical (OH), hydrogen peroxide (HO), 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; O, using a fluorescent probe; and O and HO, 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 O and HO at 100 μM. l-Dehydroascorbate (DHA), an oxidized form of Asc, degraded HO, whereas it did not quench OH or O, which are sources of HO. Furthermore, EGCG efficiently scavenged NTP-induced O, O, and HO 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.
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http://dx.doi.org/10.1016/j.abb.2021.108762DOI Listing
March 2021

Neural stem cell-specific ITPA deficiency causes neural depolarization and epilepsy.

JCI Insight 2020 11 19;5(22). Epub 2020 Nov 19.

Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, and.

Inosine triphosphate pyrophosphatase (ITPA) hydrolyzes inosine triphosphate (ITP) and other deaminated purine nucleotides to the corresponding nucleoside monophosphates. In humans, ITPA deficiency causes severe encephalopathy with epileptic seizure, microcephaly, and developmental retardation. In this study, we established neural stem cell-specific Itpa-conditional KO mice (Itpa-cKO mice) to clarify the effects of ITPA deficiency on the neural system. The Itpa-cKO mice showed growth retardation and died within 3 weeks of birth. We did not observe any microcephaly in the Itpa-cKO mice, although the female Itpa-cKO mice did show adrenal hypoplasia. The Itpa-cKO mice showed limb-clasping upon tail suspension and spontaneous and/or audiogenic seizure. Whole-cell patch-clamp recordings from entorhinal cortex neurons in brain slices revealed a depolarized resting membrane potential, increased firing, and frequent spontaneous miniature excitatory postsynaptic current and miniature inhibitory postsynaptic current in the Itpa-cKO mice compared with ITPA-proficient controls. Accumulated ITP or its metabolites, such as cyclic inosine monophosphates, or RNA containing inosines may cause membrane depolarization and hyperexcitability in neurons and induce the phenotype of ITPA-deficient mice, including seizure.
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http://dx.doi.org/10.1172/jci.insight.140229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710303PMC
November 2020

The new era for research on polyphenols and food factors.

Arch Biochem Biophys 2020 12 12;696:108678. Epub 2020 Nov 12.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.abb.2020.108678DOI Listing
December 2020

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

Cancers (Basel) 2020 Nov 10;12(11). Epub 2020 Nov 10.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-Ku, Nagoya 466-8550, Japan.

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.
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http://dx.doi.org/10.3390/cancers12113320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698037PMC
November 2020

Prognostic significance of the MDM2/HMGA2 ratio and histological tumor grade in dedifferentiated liposarcoma.

Genes Chromosomes Cancer 2021 Jan 28;60(1):26-37. Epub 2020 Oct 28.

Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Dedifferentiated liposarcoma (DDLPS) is a relatively common soft tissue sarcoma that results from the progression of well-differentiated liposarcoma (WDLPS). This study aimed to investigate the progression process and to clarify the pathological and genetic factors related to poor prognosis in DDLPS. In 32 DDLPS cases and five WDLPS cases, genetic factors were analyzed by custom comparative genomic hybridization (CGH) array, which was designed to densely cover gene regions known to be frequently amplified in WD/DDLPS. The analyses comparing primary and metastatic lesions and those comparing histologically different areas in the same tumor revealed intra-tumoral genetic heterogeneity and progression. According to a prognostic analysis comparing the good-prognosis and the poor-prognosis groups, we selected MDM2 and HMGA2 as candidate genes associated with poor and good prognosis, respectively. The ratios of the amplification or gain levels of MDM2 and HMGA2 expressed in log ratios (log[MDM2/HMGA2] = log[MDM2]-log[HMGA2]) were significantly associated with prognosis. An amplification or gain level of MDM2 that was more than twice that of HMGA2 (MDM2/HMGA2 > 2, log[MDM2/HMGA2] > 1) was strongly related to poor OS (P < .001) and poor distant metastasis-free survival (DMFS) (P < .001). In the pathological analysis of 44 cases of DDLPS, histological tumor grade, cellular atypia, and MDM2 immunoreactivity were related to overall survival (OS), while HMGA2 immunoreactivity tended to be associated with OS. Cellular atypia was also associated with DMFS. In conclusion, histological grade and MDM2 expression were determined to be prognostically important, and the MDM2/HMGA2 amplification or gain ratio was found to have significant prognostic value by the custom CGH array analysis.
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http://dx.doi.org/10.1002/gcc.22899DOI Listing
January 2021

Defective biosynthesis of ascorbic acid in Sod1-deficient mice results in lethal damage to lung tissue.

Free Radic Biol Med 2021 01 21;162:255-265. Epub 2020 Oct 21.

Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata, 990-9585, Japan.

Superoxide dismutase 1 (Sod1) plays pivotal roles in antioxidation via accelerating the conversion of superoxide anion radicals into hydrogen peroxide, thus inhibiting the subsequent radical chain reactions. While Sod1 deficient cells inevitably undergo death in culture conditions, Sod1-knockout (KO) mice show relatively mild phenotypes and live approximately two years. We hypothesized that the presence of abundant levels of ascorbic acid (AsA), which is naturally produced in mice, contributes to the elimination of reactive oxygen species (ROS) in Sod1-KO mice. To verify this hypothesis, we employed mice with a genetic ablation of aldehyde reductase (Akr1a), an enzyme that is involved in the biosynthesis of AsA, and established double knockout (DKO) mice that lack both Sod1 and Akr1a. Supplementation of AsA (1.5 mg/ml in drinking water) was required for the DKO mice to breed, and, upon terminating the AsA supplementation, they died within approximately two weeks regardless of age or gender. We explored the etiology of the death from pathophysiological standpoints in principal organs of the mice. Marked changes were observed in the lungs in the form of macroscopic damage after the AsA withdrawal. Histological and immunological analyses of the lungs indicated oxidative damage of tissue and activated immune responses. Thus, preferential oxidative injury that occurred in pulmonary tissues appeared to be primary cause of the death in the mice. These collective results suggest that the pivotal function of AsA in coping with ROS in vivo, is largely in pulmonary tissues that are exposed to a hyperoxygenic microenvironment.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.10.023DOI Listing
January 2021

Novel ovarian endometriosis model causes infertility via iron-mediated oxidative stress in mice.

Redox Biol 2020 10 15;37:101726. Epub 2020 Sep 15.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-Ku, Nagoya, 466-8550, Japan; Center for Low Temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya, 484-8601, Japan; Sydney Medical School, The University of Sydney, NSW, Australia. Electronic address:

Ovarian endometriosis (OE) provides women of reproductive age with not only severe menstrual pain but also infertility and an increased risk for ovarian carcinogenesis. Whereas peritoneal endometriosis models have been developed with syngeneic implantation of minced uterine tissue and oncogenic K-ras allele with conditional Pten deletion within ovarian surface epithelium generated preneoplastic endometrial glandular morphology, followed by endometrioid adenocarcinoma, there has been no mouse model of OE similar to human counterparts, applicable to preclinical studies. Here we for the first time established a murine OE model that reveals infertility, and evaluated the involvement of iron catalyzed oxidative stress in the pathogenesis. Minced uterine tissue from female mice was implanted on ovarian surface of syngeneic mice after bursectomy to induce OE. Ectopic growth of endometrium was observed in association with ovary 4 weeks after implantation in 85.7% (12/14) of the operated mice with our protocol. Endometriotic lesions involved intestine, pancreas and peritoneal wall. Fibrosis around the ovary was prominent and increased time-dependently in the OE group. Iron accumulation was significantly increased in the OE group, leading to oxidative stress in each stage of the follicles as evaluated by 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2'-deoxyguanosine. Expression of follicle stimulating hormone receptor in the follicles revealed a significant decrease during pre-antral, antral and pre-ovulatory phases in the OE group. Finally, the number of pups was significantly reduced in the OE group in comparison to the controls. This model affords an opportunity to evaluate agents or procedures to counteract ovarian endometriosis in the preclinical settings.
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http://dx.doi.org/10.1016/j.redox.2020.101726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509075PMC
October 2020

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

Redox Biol 2020 09 24;36:101616. Epub 2020 Jun 24.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan; Sydney Medical School, The University of Sydney, NSW, 2006, Australia. Electronic address:

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 β-catenin activation. Conversely, β-catenin overexpression in mesothelial cells induced higher intracellular catalytic Fe(II) with increased G2/M cell-cycle fraction, when p16 genomic loci localized more peripherally in the nucleus. Mesothelial cells after challenge of HO under β-catenin overexpression presented low p16 expression with a high incidence of deletion in p16 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.
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http://dx.doi.org/10.1016/j.redox.2020.101616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330611PMC
September 2020

Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice.

Cancer Sci 2020 Oct 21;111(10):3576-3587. Epub 2020 Aug 21.

Division of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Japan.

Cervical cancer (CC) is usually initiated by infection with high-risk types of human papillomavirus (HPV). The HPV E6 and E7 proteins target p53 and RB, respectively, but other cellular targets likely exist. We generated uterus-specific MOB1A/B double KO (uMob1DKO) mice, which immediately developed cervical squamous cell carcinoma in situ. Mutant cervical epithelial cells showed YAP1-dependent hyperproliferation, altered self-renewal, impaired contact inhibition, and chromosomal instability. p53 activation was increased in uMob1DKO cells, and additional p53 loss in uMob1DKO mice accelerated tumor invasion. In human CC, strong YAP1 activation was observed from the precancerous stage. Human cells overexpressing HPV16 E6/E7 showed inactivation of not only p53 and RB but also PTPN14, boosting YAP1 activation. Estrogen, cigarette smoke condensate, and PI3K hyperactivation all increased YAP1 activity in human cervical epithelial cells, and PTPN14 depletion along with PI3K activation or estrogen treatment further enhanced YAP1. Thus, immediate CC onset may initiate when YAP1 activity exceeds an oncogenic threshold, making Hippo-YAP1 signaling a major CC driver.
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http://dx.doi.org/10.1111/cas.14581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541006PMC
October 2020

Connective tissue growth factor produced by cancer‑associated fibroblasts correlates with poor prognosis in epithelioid malignant pleural mesothelioma.

Oncol Rep 2020 Sep 3;44(3):838-848. Epub 2020 Jul 3.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya 466‑8550, Japan.

Malignant mesothelioma is an aggressive neoplasm for which effective treatments are lacking. We often encounter mesothelioma cases with a profound desmoplastic reaction, suggesting the involvement of cancer‑associated fibroblasts (CAFs) in mesothelioma progression. While the roles of CAFs have been extensively studied in other tumors and have led to the view that the cancer stroma contains heterogeneous populations of CAFs, their roles in mesothelioma remain unknown. We previously showed that connective tissue growth factor (CTGF), a secreted protein, is produced by both mesothelioma cells and fibroblasts and promotes the invasion of mesothelioma cells in vitro. In this study, we examined the clinical relevance of CAFs in mesothelioma. Using surgical specimens of epithelioid malignant pleural mesothelioma, we evaluated the clinicopathological significance of the expression of α‑smooth muscle actin (αSMA), the most widely used marker of CAFs, the expression of CTGF, and the extent of fibrosis by immunohistochemistry and Elastica‑Masson staining. We also analyzed the expression of mesenchymal stromal cell‑ and fibroblast‑expressing Linx paralogue (Meflin; ISLR), a recently reported CAF marker that labels cancer‑restraining CAFs and differ from αSMA‑positive CAFs, by in situ hybridization. The extent of fibrosis and CTGF expression in mesothelioma cells did not correlate with patient prognosis. However, the expression of αSMA and CTGF, but not Meflin, in CAFs correlated with poor prognosis. The data suggest that CTGF+ CAFs are involved in mesothelioma progression and represent a potential molecular target for mesothelioma therapy.
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http://dx.doi.org/10.3892/or.2020.7669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388423PMC
September 2020

The new role of poly (rC)-binding proteins as iron transport chaperones: Proteins that could couple with inter-organelle interactions to safely traffic iron.

Biochim Biophys Acta Gen Subj 2020 11 15;1864(11):129685. Epub 2020 Jul 15.

Kenjinkai Healthcare Corporation, Japan. Electronic address:

Background: Intracellular iron transport is mediated by iron chaperone proteins known as the poly(rC)-binding proteins (PCBPs), which were originally identified as RNA/DNA-binding molecules.

Scope Of Review: PCBPs assume a role as not only as cytosolic iron carriers, but also as regulators of iron transport and recycling. PCBP1 is involved in the iron storage pathway that involves ferritin, while PCBP2 is involved in processes that include: iron transfer from the iron importer, divalent metal ion transporter 1; iron export mediated by ferroportin-1; and heme degradation via heme oxygenase 1.

Major Conclusions: Both PCBP1 and PCBP2 possess iron-binding activity and form hetero/homo dimer complexes. These iron chaperones have a subset of non-redundant functions and regulate iron metabolism independently.

General Significance: This intracellular iron chaperone system mediated by PCBPs provide a transport "gateway" of ferrous iron that may potentially link with dynamic, inter-organelle interactions to safely traffic intracellular iron.
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http://dx.doi.org/10.1016/j.bbagen.2020.129685DOI Listing
November 2020

Role of carbonic anhydrases in ferroptosis-resistance.

Arch Biochem Biophys 2020 08 30;689:108440. Epub 2020 May 30.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan; Sydney Medical School, The University of Sydney, NSW, Australia. Electronic address:

Iron is essential for all the lives on earth but may trigger a switch toward ferroptosis, a novel form of regulated necrosis. Carbonic anhydrases (CAs) are ubiquitous enzymes from microbes to humans. The primary function of CAs is to regulate cellular pH by hydrating carbon dioxide (CO) to protons (H) and bicarbonate ions (HCO). Furthermore, CAs play roles in biosynthetic reactions, such as gluconeogenesis, lipogenesis, ureagenesis and are also associated with tumor metabolism, suggesting that CAs may be a potential target for the treatment of cancers. We have recently revealed a novel function of CA IX in ferroptosis-resistance by using human malignant mesothelioma cells. Herein, we aim to review the potential molecular association between ferroptosis and CAs, from the viewpoint of iron-metabolism, lipogenesis and signaling pathways both under physiological and pathological contexts.
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http://dx.doi.org/10.1016/j.abb.2020.108440DOI Listing
August 2020

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

Arch Biochem Biophys 2020 07 25;688:108414. Epub 2020 May 25.

Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya, 484-8601, Japan.

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.
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http://dx.doi.org/10.1016/j.abb.2020.108414DOI Listing
July 2020

The new era for redox research.

Free Radic Res 2020 Dec;54(11-12):787-789

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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http://dx.doi.org/10.1080/10715762.2020.1774177DOI Listing
December 2020

Ferroptosis at the crossroads of infection, aging and cancer.

Cancer Sci 2020 Aug 20;111(8):2665-2671. Epub 2020 Jun 20.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Despite significant developments and persistent efforts by scientists, cancer is one of the primary causes of human death worldwide. No form of life on Earth can survive without iron, although some species can live without oxygen. Iron presents a double-edged sword. Excess iron is a risk for carcinogenesis, while its deficiency causes anemia, leading to oxygen shortage. Every cell is eventually destined to death, either through apoptosis or necrosis. Regulated necrosis is recognized in distinct forms. Ferroptosis is defined as catalytic Fe(II)-dependent regulated necrosis accompanied by lipid peroxidation. The main observation was necrosis of fibrosarcoma cells through inhibition of cystine/glutamate antiporter with erastin, which reduced intracellular cysteine and, thus, glutathione levels. Our current understanding of ferroptosis is relative abundance of iron (catalytic Fe[II]) in comparison with sulfur (sulfhydryls). Thus, either excess iron or sulfur deficiency causes ferroptosis. Cell proliferation inevitably requires iron for DNA synthesis and energy production. Carcinogenesis is a process toward iron addiction with ferroptosis resistance. Conversely, ferroptosis is associated with aging and neurodegeneration. Ferroptosis of immune cells during infection is advantageous for infectious agents, whereas ferroptosis resistance incubates carcinogenic soil as excess iron. Cancer cells are rich in catalytic Fe(II). Directing established cancer cells to ferroptosis is a novel strategy for discovering cancer therapies. Appropriate iron regulation could be a tactic to reduce and delay carcinogenesis.
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http://dx.doi.org/10.1111/cas.14496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419040PMC
August 2020

Augmented oxidative stress increases 8-oxoguanine preferentially in the transcriptionally active genomic regions.

Free Radic Res 2020 Dec 16;54(11-12):872-882. Epub 2020 Apr 16.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

8-Oxoguanine (8-oxoG) is the most common DNA base modification in the mammalian genome, associated with oxidative stress. Here we analysed the alterations in the distribution of 8-oxoG across the entire murine genome, before and after an elevation of oxidative stress by the use of ferric nitrilotriacetate (Fe-NTA) as an oxidative stress inducer in the renal proximal tubules. We isolated DNA fragments containing 8-oxoGs with immunoprecipitation from the murine genome, and amplified them by PCR for a distribution analysis with microarray-based comparative genomic hybridisation. The distribution profiles revealed that frequencies of 8-oxoG fluctuated with a cycle of 1-10 Mb along the chromosomes and the amplitude of the fluctuation was reduced after Fe-NTA administration. The distributions of 8-oxoG along the entire genome in the control and oxidatively stressed conditions were negatively correlated with that of gene density but positively correlated with that of Lamin B1 interaction, which corresponds to lamina-associated domains. These results on the murine genome were consistent with those on the rat genome we previously reported. We further discovered a negative correlation between the distributions of 8-oxoG and transcriptional activity along the genome. Finally, a comparison of the distributions before and after Fe-NTA administration suggested that 8-oxoGs are generated in response to the augmented oxidative stress preferentially in the transcriptionally active genomic regions, where 8-oxoGs have been less accumulated in the control condition.
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http://dx.doi.org/10.1080/10715762.2020.1733548DOI Listing
December 2020

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

Cancer Sci 2020 Jun 5;111(6):2016-2027. Epub 2020 May 5.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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.
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http://dx.doi.org/10.1111/cas.14405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293088PMC
June 2020

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

Free Radic Res 2020 Mar;54(2-3):195-205

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.

Exposure to asbestos fiber is central to mesothelial carcinogenesis. Recent sequencing studies on human and rodent malignant mesothelioma (MM) revealed frequently mutated genes, including , and . 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 and . 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 KO, 41.4% (12/29) in KO and 24.0% (6/25) in KO mice, whereas 31.7% (20/63) induction was observed in wild-type (Wt) mice. The lifespan of female KO mice was longer than that of female Wt mice ( = 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.
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http://dx.doi.org/10.1080/10715762.2020.1743285DOI Listing
March 2020

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

Cancer Sci 2020 Apr 21;111(4):1180-1192. Epub 2020 Mar 21.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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.
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http://dx.doi.org/10.1111/cas.14358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156836PMC
April 2020

Plasma-activated medium promotes autophagic cell death along with alteration of the mTOR pathway.

Sci Rep 2020 01 31;10(1):1614. Epub 2020 Jan 31.

Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

The biological function of non-thermal atmospheric pressure plasma has been widely accepted in several types of cancer. We previously developed plasma-activated medium (PAM) for clinical use, and demonstrated that PAM exhibits a metastasis-inhibitory effect on ovarian cancer through reduced MMP-9 secretion. However, the anti-tumor effects of PAM on endometrial cancer remain unknown. In this study, we investigated the inhibitory effect of PAM on endometrial cancer cell viability in vitro. Our results demonstrated that AMEC and HEC50 cell viabilities were reduced by PAM at a certain PAM ratio, and PAM treatment effectively increased autophagic cell death in a concentration dependent manner. In addition, we evaluated the molecular mechanism of PAM activity and found that the mTOR pathway was inactivated by PAM. Moreover, our results demonstrated that the autophagy inhibitor MHY1485 partially inhibited the autophagic cell death induced by PAM treatment. These findings indicate that PAM decreases the viability of endometrial cancer cells along with alteration of the mTOR pathway, which is critical for cancer cell viability. Collectively, our data suggest that PAM inhibits cell viability while inducing autophagic cell death in endometrial cancer cells, representing a potential novel treatment for endometrial cancer.
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http://dx.doi.org/10.1038/s41598-020-58667-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994502PMC
January 2020

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

Sci Rep 2019 09 20;9(1):13657. Epub 2019 Sep 20.

Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan.

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.
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http://dx.doi.org/10.1038/s41598-019-50136-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754505PMC
September 2019

Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia.

Redox Biol 2019 09 10;26:101297. Epub 2019 Aug 10.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan; Sydney Medical School, The University of Sydney, NSW, Australia. Electronic address:

Hypoxia and acidity provide microenvironment for selection under evolutionary pressure and proliferation in cancer cells. Carbonic anhydrases (CAs) are a superfamily of metalloenzymes present in all life kingdoms, equilibrating the reactions among CO, bicarbonate and H. CA9, a membrane-associated α-CA, has been a drug target for various cancers. Whereas iron is essential not only for cancer cells but also for all the lives on earth, little is known on the association among hypoxia, iron metabolism, extracellular acidity and redox regulation. Malignant mesothelioma (MM), an aggressive tumor with poor prognosis, is an intriguing model in that asbestos-associated pathogenesis includes excess iron environment during carcinogenesis. Re-analysis of rat asbestos-induced MM model revealed an inverse association between high CA9 expression and survival. Here we used human MMs to identify the molecular events surrounding CA9 from the viewpoint of iron metabolism. CA9 expression was significantly higher in MM cells than in MeT-5A mesothelial cells, which was further amplified under hypoxia (1%O) with increased catalytic Fe(II). CA9 suppression by inhibitors (S4 and U104) decreased viability and migration of MM cells, accompanied by overexpression of TFRC, IREB1/2 and FPN1(SLC40A1) and by downregulation of FTH/FTL. This expressional pattern was similar to that of erastin-induced ferroptosis in the same cells. Furthermore, we observed mitochondrial fission and enhanced autophagy with increased catalytic Fe(II) in both mitochondria and lysosomes after CA9 inhibition, accompanied by increased peroxides, mitochondrial O and lipid peroxidation. The eventual cell death was significantly inhibited by deferoxamine, ferrostatin-1 and Z-VAD-FMK, suggesting a mixed cell death of ferroptosis and apoptosis. Therefore, CA9 plays a role in equilibrating among hypoxia, iron metabolism and redox regulation in MM cells.
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http://dx.doi.org/10.1016/j.redox.2019.101297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831888PMC
September 2019

How iron is handled in the course of heme catabolism: Integration of heme oxygenase with intracellular iron transport mechanisms mediated by poly (rC)-binding protein-2.

Arch Biochem Biophys 2019 09 14;672:108071. Epub 2019 Aug 14.

Kenjinkai Healthcare Corporation, 530 Asa, Sanyo-Onoda Yamaguchi, 757-0001, Japan. Electronic address:

Heme and iron are essential to almost all forms of life. The strict maintenance of heme and iron homeostasis is essential to prevent cellular toxicity and the existence of systemic and intracellular regulation is fundamental. Cytosolic heme can be catabolized and detoxified by heme oxygenases (HOs). Interestingly, free heme detoxification through HOs results in the production of free ferrous iron, which can be potentially hazardous for cells. Recently, the intracellular iron chaperone, poly (rC)-binding protein 2 (PCBP2), has been identified, which can be involved in accepting iron after heme catabolism as well as intracellular iron transport. In fact, HO1, NADPH-cytochrome P450 reductase, and PCBP2 form a functional unit that integrates the catabolism of heme with the binding and transport of iron by PCBP2. In this review, we provide an overview of our understanding of the iron chaperones and discuss the mechanism how iron chaperones bind iron released during the process of heme degradation.
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http://dx.doi.org/10.1016/j.abb.2019.108071DOI Listing
September 2019

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

J Clin Biochem Nutr 2019 Jul 1;65(1):8-15. Epub 2019 Jun 1.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Turumai-cho, Showa-ku, Nagoya 466-8550, Japan.

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.
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http://dx.doi.org/10.3164/jcbn.18-91DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667380PMC
July 2019

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

Arch Biochem Biophys 2019 07 31;669:87-95. Epub 2019 May 31.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.abb.2019.05.019DOI Listing
July 2019