Faculty Profiles - TOYOKUNI Shinya

RhoA and vigilin are candidates for immunohistochemical markers for epithelioid malignant mesothelioma.

Hiratsuka T, Yamamoto T, Yoshizawa A, Toyokuni S, Tsuruyama T

Scientific reports Vol. 12 ( 1 ) page： 18519 2022.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Reports

Diagnostic markers of malignant mesothelioma (MM) have been extensively investigated. Immunohistochemistry (IHC) markers, such as calretinin, have been used for pathologic diagnosis. However, more diagnostic markers are required to improve the specificity and sensitivity of pathologic diagnosis. This study proposed two proteins as diagnostic markers for epithelioid MM. One is RhoA, an MM mutation-susceptible locus-derived protein, and another is vigilin, a lung small cell carcinoma marker. IHC was performed using 93 MM (epithelioid, 71 cases; sarcomatoid, 13 cases; and biphasic, 9 cases), 64 lung adenocarcinoma (LAC), 60 lung squamous cell carcinoma (LSC), and 14 normal mesothelial (NM) tissues. The majority of epithelioid MM cases were positive for both RhoA and vigilin, whereas both IHCs showed lower stainability in biphasic and sarcomatoid MM. Besides, both IHCs showed significantly higher stainability for RhoA and vigilin in epithelioid MM than in LAC and LSC (p < 0.05). Chi-square tests showed that both RhoA and vigilin IHC positive rate in epithelioid MM was not significantly different from that of calretinin (p > 0.05). In the differential diagnosis of MM from lung cancer, the accuracy and specificity of RhoA, vigilin, and calretinin staining were almost equivalent. Further, H-score test showed that there was no significant difference between RhoA versus calretinin and vigilin versus calretinin in IHC positivity in epithelioid MM (p > 0.05). In conclusion, RhoA and vigilin may be candidates for immunohistochemical markers for epithelioid MM.

DOI： 10.1038/s41598-022-20334-0

Molecular hydrogen has a positive impact on pregnancy maintenance through enhancement of mitochondrial function and immunomodulatory effects on T cells. International journal

Chieko Aoki, Kenji Imai, Teruyuki Mizutani, Daisuke Sugiyama, Rika Miki, Yoshihiro Koya, Tomoko Kobayashi, Takafumi Ushida, Yukako Iitani, Noriyuki Nakamura, Taro Owaki, Hiroyoshi Nishikawa, Shinya Toyokuni, Hiroaki Kajiyama, Tomomi Kotani

Life sciences Vol. 308 page： 120955 - 120955 2022.11

Language：English Publishing type：Research paper (scientific journal)

AIMS: Molecular hydrogen (H2) has attracted growing interest because of its implications in various diseases. However, the molecular mechanisms underlying the remarkable effect of a small amount of H2 remain elusive. No knowledge has been available on the role of H2 in the etiology of pregnancy disorders or its direct influence on human immune cells. Since maternal immunity, T cells in particular, plays a critical role in pregnancy maintenance. We investigated the effects of H2 on T cells and its relation to preterm birth (PTB). MAIN METHODS: Exhaled H2 concentrations in pregnant women were measured and correlated with cytokine concentrations in maternal and umbilical cord blood. H2 was added to T cells collected from healthy donors, and differentiation and proliferation were examined. Energy metabolism was also examined. H2 was administered to mice and cytokine expression was compared. KEY FINDINGS: Our prospective observational study revealed that maternal production of H2 is significantly lower in pregnant women with PTB, suggesting its potential as a biomarker for predicting PTB. We found that H2 has clear associations with several maternal cytokines, and acts as an immunomodulator by exerting mitochondrial function in human T cells. Moreover, in vivo administration of H2 to pregnant mice regulated inflammatory responses and reduced PTB caused by T cell activation, which further supports the notion that H2 may contribute to prolonged gestation through its immunomodulatory effect. SIGNIFICANCE: Measuring maternal H2-production could be a potential clinical tool in the management of PTB, and H2 may have positive impact on pregnancy maintenance.

DOI： 10.1016/j.lfs.2022.120955

Commentary for an article on photooxidation in isolated chloroplasts. International journal

Shinya Toyokuni, Yingyi Kong, Danyang Mi

Archives of biochemistry and biophysics Vol. 726 page： 109133 - 109133 2022.9

Language：English Publishing type：Research paper (scientific journal)

This commentary concerns a highly cited paper by Robert L Heath and Lester Packer in Archives of Biochemistry and Biophysics published in 1968. Chloroplasts are organelles in algae and plants that use light energy for carbon fixation and oxygen production. These authors discovered that isolated chloroplasts exposed to visible light undergo a cyclic peroxidation of tri-unsaturated fatty acids, contributing to the double-edged sword concept of electron transfer reactions.

DOI： 10.1016/j.abb.2022.109133

Iron as spirit of life to share under monopoly.

Shinya Toyokuni, Yingyi Kong, Hao Zheng, Yuki Maeda, Yashiro Motooka, Shinya Akatsuka

Journal of clinical biochemistry and nutrition Vol. 71 ( 2 ) page： 78 - 88 2022.9

Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

Any independent life requires iron to survive. Whereas iron deficiency causes oxygen insufficiency, excess iron is a risk for cancer, generating a double-edged sword. Iron metabolism is strictly regulated via specific systems, including iron-responsive element (IRE)/iron regulatory proteins (IRPs) and the corresponding ubiquitin ligase FBXL5. Here we briefly reflect the history of bioiron research and describe major recent advancements. Ferroptosis, a newly coined Fe(II)-dependent regulated necrosis, is providing huge impact on science. Carcinogenesis is a process to acquire ferroptosis-resistance and ferroptosis is preferred in cancer therapy due to immunogenicity. Poly(rC)-binding proteins 1/2 (PCBP1/2) were identified as major cytosolic Fe(II) chaperone proteins. The mechanism how cells retrieve stored iron in ferritin cores was unraveled as ferritinophagy, a form of autophagy. Of note, ferroptosis may exploit ferritinophagy during the progression. Recently, we discovered that cellular ferritin secretion is through extracellular vesicles (EVs) escorted by CD63 under the regulation of IRE/IRP system. Furthermore, this process was abused in asbestos-induced mesothelial carcinogenesis. In summary, cellular iron metabolism is tightly regulated by multi-system organizations as surplus iron is shared through ferritin in EVs among neighbor and distant cells in need. However, various noxious stimuli dramatically promote cellular iron uptake/storage, which may result in ferroptosis.

DOI： 10.3164/jcbn.22-43

Association of alcohol intake and female gender with high expression of TMPRSS2 in tongue as potential risk for SARS-CoV-2 infection

Kotaro Sato, Koki Fujii, Noriyuki Yamamoto, Norihisa Ichimura, Satoshi Yamaguchi, Hirohisa Yamada, Hideharu Hibi, Shinya Toyokuni

Journal of Clinical Biochemistry and Nutrition Vol. 71 ( 2 ) page： 129 - 135 2022.9

Language：English Publishing type：Research paper (scientific journal) Publisher：The Society for Free Radical Research Japan

COVID-19 is pandemic since 2020 and further information is necessary on the risk factors associated with the infection of SARS-CoV-2. As an entry mechanism, SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as receptor and transmembrane serine protease 2 (TMPRSS2) to activate fusion with host plasma membrane. Because dysgeusia is an early symptom of COVID-19, we here studied the expression of ACE2 and TMPRSS2 in the tongue and the associated tissues of mice and humans with immunohistochemistry and immunoblot analysis. ACE2 expression was low in the human tongue but was observed in the squamous epithelium, perineurium, arterial wall, salivary glands as well as taste buds. In contrast, mice showed high expression. In sharp contrast, TMPRSS2 expression was high in all the cells mentioned above in humans but relatively low in mice except for salivary glands. We then performed semi-quantitation of immunohistochemistry data of human ACE2 and TMPRSS2 and analyzed for age, sex, alcohol intake, and smoking habit with logistic regression analysis. We found that alcohol intake and female gender were the significant risk factors for increasing TMPRSS2 expression. In conclusion, TMPRSS2 is an important factor to be considered regarding SARS-CoV-2 entry and amplification in the oral cavity, which is promoted through drinking habit.

DOI： 10.3164/jcbn.21-172

環境因子であるタルクおよびアスベストの卵巣発がん性とゲノム毒性の検討(Carcinogenic genome toxicity of environmental factors, asbestos and talc in ovary)

本岡大社, 鬼丸洸, 鈴木洋, 片渕充沙子, 近藤英治, 田代浩徳, 片渕秀隆, 豊國伸哉

日本癌学会総会記事 Vol. 81回 page： P - 2007 2022.9

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

Okazaki Y, Ito N, Tanaka H, Hori M, Toyokuni S

Free radical research Vol. 56 ( 9-10 ) page： 595 - 606 2022.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Free Radical Research

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.

DOI： 10.1080/10715762.2022.2157272

BRCA1のハプロ不全は発がん過程において、フェロトーシス抵抗性を獲得することにより染色体増幅を促進する(BRCA1 haploinsufficiency promotes carcinogenic chromosomal amplification by ferroptosis-resistance)

プラズマ活性化された乳酸リンゲルに誘導されたフェロトーシスにおけるpcbp1/2とグルタチオンとの協同作用(Cooperation of Poly(rC)-binding Proteins 1/2 and Glutathione in Ferroptosis Induced by Plasma-activated Ringer's Lactate)

鄭好, 蒋麗, 呂沁穎, 赤塚慎也, 本岡大社, 関戸好孝, 中村香江, 田中宏昌, 石川健治, 梶山広明, 水野正明, 堀勝, 豊國伸哉

日本癌学会総会記事 Vol. 81回 page： E - 2004 2022.9

個別化予防医療・先制医療 BRCA1のハプロ不全は、腎臓発がん過程において、フェロトーシス抵抗性を獲得することにより染色体増幅を促進する(BRCA1 haploinsufficiency promotes chromosomal amplification under renal carcinogenesis through ferroptosis-resistance)

孔穎怡, 赤塚慎也, 本岡大社, 鄭好, 白木之浩, 真下知士, 今岡達彦, 豊國伸哉

日本癌学会総会記事 Vol. 81回 page： S20 - 7 2022.9

孔穎怡, 赤塚慎也, 本岡大社, 鄭好, 白木之浩, 真下知士, 今岡達彦, 豊國伸哉

Biomedical Research on Trace Elements Vol. 33 ( 1 ) page： 130 - 130 2022.9

BRCA1遺伝子ハプロ不全モデルラットを用いた卵巣予備能低下の検証(Validation of reduced ovarian reserve in BRCA1 haploinsufficient rats.)

曽根原玲菜, 本岡大社, 梶山広明, 真下知士, 今岡達彦, 豊國伸哉

日本癌学会総会記事 Vol. 81回 page： P - 3012 2022.9

BRCA1欠損はフェロトーシス抵抗性を誘導することによりアスベスト誘発性腹膜中皮腫を促進する(Ferroptosis resistance is induced by BRCA1 deficiency in asbestos-induced mesothelioma)

羅亜光, 本岡大社, 赤塚慎也, 真下知士, 今岡達彦, 豊國伸哉

日本癌学会総会記事 Vol. 81回 page： P - 2003 2022.9

環境因子であるタルクおよびアスベストの卵巣発がん性とゲノム毒性の検討(Carcinogenic genome toxicity of environmental factors, asbestos and talc in ovary)

本岡大社, 鬼丸洸, 鈴木洋, 片渕充沙子, 近藤英治, 田代浩徳, 片渕秀隆, 豊國伸哉

日本癌学会総会記事 Vol. 81回 page： P - 2007 2022.9

BRCA1 haploinsufficiency promotes chromosomal amplification under Fenton reaction-based carcinogenesis through ferroptosis-resistance. International journal

Yingyi Kong, Shinya Akatsuka, Yashiro Motooka, Hao Zheng, Zhen Cheng, Yukihiro Shiraki, Tomoji Mashimo, Tatsuhiko Imaoka, Shinya Toyokuni

Redox biology Vol. 54 page： 102356 - 102356 2022.8

Language：English Publishing type：Research paper (scientific journal)

Germline-mutation in BRCA1 tumor suppressor gene is an established risk for carcinogenesis not only in females but also in males. Deficiency in the repair of DNA double-strand breaks is hypothesized as a responsible mechanism for carcinogenesis. However, supporting data is insufficient both in the mutation spectra of cancers in the patients with BRCA1 germline-mutation and in murine knockout/knock-in models of Brca1 haploinsufficiency. Furthermore, information on the driving force toward carcinogenesis in BRCA1 mutation carriers is lacking. Here we applied Fenton reaction-based renal carcinogenesis to a rat heterozygously knockout model of BRCA1 haploinsufficiency (mutant [MUT] model; L63X/+). Rat MUT model revealed significant promotion of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Array-based comparative genome hybridization of the RCCs identified significant increase in chromosomal amplification, syntenic to those in breast cancers of BRCA1 mutation carriers, including c-Myc, in comparison to those in the wild-type. Subacute-phase analysis of the kidney after repeated Fe-NTA treatment in the MUT model revealed dysregulated iron metabolism with mitochondrial malfunction assessed by expression microarray and electron microscopy, leading to renal tubular proliferation with iron overload. In conclusion, we for the first time demonstrate that biallelic wild-type BRCA1 provides more robust protection for mitochondrial metabolism under iron-catalyzed oxidative stress, preventing the emergence of neoplastic cells with chromosomal amplification. Our results suggest that oxidative stress via excess iron is a major driving force for carcinogenesis in BRCA1 haploinsufficiency, which can be a target for cancer prevention and therapeutics.

DOI： 10.1016/j.redox.2022.102356

Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers

Hirotoshi Soyama, Miki Nishio, Junji Otani, Toshiko Sakuma, Shintaro Takao, Shigeo Hara, Takaaki Masuda, Koshi Mimori, Shinya Toyokuni, John P. Lydon, Kazuwa Nakao, Hiroshi Nishina, Takumi Fukumoto, Tomohiko Maehama, Akira Suzuki

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vol. 119 ( 29 ) page： e2123134119 2022.7

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

Breast cancer is the most frequent malignancy in women worldwide. Basal-like breast cancer (BLBC) is the most aggressive form of this disease, and patients have a poor prognosis. Here, we present data suggesting that the Hippo-transcriptional coactivator with PDZ-binding motif (TAZ) pathway is a key driver of BLBC onset and progression. Deletion of Mob1a/b in mouse mammary luminal epithelium induced rapid and highly reproducible mammary tumorigenesis that was dependent on TAZ but not yes-associated protein 1 (YAP1). In situ early-stage BLBC-like malignancies developed in mutant animals by 2 wk of age, and invasive BLBC appeared by 4 wk. In a human estrogen receptor(+) luminal breast cancer cell line, TAZ hyperactivation skewed the features of these luminal cells to the basal phenotype, consistent with the aberrant TAZ activation frequently observed in human precancerous BLBC lesions. TP53 mutation is rare in human precancerous BLBC but frequent in invasive BLBC. Addition of Trp53 deficiency to our Mob1a/b-deficient mouse model enhanced tumor grade and accelerated cancer progression. Our work justifies targeting the Hippo-TAZ pathway as a therapy for human BLBC, and our mouse model represents a powerful tool for evaluating candidate agents.

DOI： 10.1073/pnas.2123134119

Hepatocyte growth factor derived from senescent cells attenuates cell competition-induced apical elimination of oncogenic cells. International journal

Nanase Igarashi, Kenichi Miyata, Tze Mun Loo, Masatomo Chiba, Aki Hanyu, Mika Nishio, Hiroko Kawasaki, Hao Zheng, Shinya Toyokuni, Shunsuke Kon, Keiji Moriyama, Yasuyuki Fujita, Akiko Takahashi

Nature communications Vol. 13 ( 1 ) page： 4157 - 4157 2022.7

Language：English Publishing type：Research paper (scientific journal)

Cellular senescence and cell competition are important tumor suppression mechanisms that restrain cells with oncogenic mutations at the initial stage of cancer development. However, the link between cellular senescence and cell competition remains unclear. Senescent cells accumulated during the in vivo aging process contribute toward age-related cancers via the development of senescence-associated secretory phenotype (SASP). Here, we report that hepatocyte growth factor (HGF), a SASP factor, inhibits apical extrusion and promotes basal protrusion of Ras-mutated cells in the cell competition assay. Additionally, cellular senescence induced by a high-fat diet promotes the survival of cells with oncogenic mutations, whereas crizotinib, an inhibitor of HGF signaling, provokes the removal of mutated cells from mouse livers and intestines. Our study provides evidence that cellular senescence inhibits cell competition-mediated elimination of oncogenic cells through HGF signaling, suggesting that it may lead to cancer incidence during aging.

DOI： 10.1038/s41467-022-31642-4

Diluted aqueous extract of heat-not-burn tobacco product smoke causes less oxidative damage in fibroblasts than conventional cigarette

Qinying Lyu, Li Jiang, Hao Zheng, Shotaro Hayashi, Kotaro Sato, Shinya Toyokuni

Journal of Clinical Biochemistry and Nutrition Vol. 71 ( 1 ) page： 55 - 63 2022.7

Language：English Publishing type：Research paper (scientific journal) Publisher：The Society for Free Radical Research Japan

Smoke from conventional cigarettes (C-cigarettes) contains various reactive oxygen species and toxic chemicals, which potentially cause oxidative damage not only to airways but also to the whole body, leading eventually to diseases, including emphysema, advanced atherosclerosis, and cancer. Many heat-not-burn tobacco products (HTPs) have been commercialized recently in Japan to maintain the smoking population by advertising that HTPs are less toxic. However, there were few studies reported from neutral organizations whether HTPs are indeed less damaging. To evaluate the potential capacity of HTPs to induce oxidative stress, we here compared two different HTPs with two types of C-cigarettes, using human fibroblast IMR90SV cells and 5% aqueous extracts in 10-ml phosphate-buffered saline (50-ml smoke/10 s). HTPs exhibited significantly lower oxidative toxicity in comparison to C-cigarettes. Whereas C-cigarettes induced ferroptosis in fibroblasts, the effects of HTPs were significantly reduced by measuring the levels of peroxides, pro-inflammatory cytokine expression, autophagy, catalytic Fe(II) and 8-hydroxy-2'-deoxyguanosine. Notably, major portions of C-cigarettes-induced pathogenic responses were inhibited by catalase. However, HTPs still induced p62 autophagy-adaptor at 5%-dilution and caused lethal effects to fibroblasts with undiluted solution. In conclusion, HTPs smoke per se can be toxic despite less toxicity in comparison to C-cigarettes, which warrants further investigation.

DOI： 10.3164/jcbn.21-134

Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo.

Murphy MP, Bayir H, Belousov V, Chang CJ, Davies KJA, Davies MJ, Dick TP, Finkel T, Forman HJ, Janssen-Heininger Y, Gems D, Kagan VE, Kalyanaraman B, Larsson NG, Milne GL, Nyström T, Poulsen HE, Radi R, Van Remmen H, Schumacker PT, Thornalley PJ, Toyokuni S, Winterbourn CC, Yin H, Halliwell B

Nature metabolism Vol. 4 ( 6 ) page： 651 - 662 2022.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Metabolism

Multiple roles of reactive oxygen species (ROS) and their consequences for health and disease are emerging throughout biological sciences. This development has led researchers unfamiliar with the complexities of ROS and their reactions to employ commercial kits and probes to measure ROS and oxidative damage inappropriately, treating ROS (a generic abbreviation) as if it were a discrete molecular entity. Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations. This can lead to misleading claims entering the literature and impeding progress, despite a well-established body of knowledge on how best to assess individual ROS, their reactions, role as signalling molecules and the oxidative damage that they can cause. In this consensus statement we illuminate problems that can arise with many commonly used approaches for measurement of ROS and oxidative damage, and propose guidelines for best practice. We hope that these strategies will be useful to those who find their research requiring assessment of ROS, oxidative damage and redox signalling in cells and in vivo.

DOI： 10.1038/s42255-022-00591-z

Cytotoxicity of plasma-irradiated lactate solution produced under atmospheric airtight conditions and generation of the methyl amino group

Daiki Ito, Naoyuki Iwata, Kenji Ishikawa, Kae Nakamura, Hiroshi Hashizume, Camelia Miron, Hiromasa Tanaka, Hiroaki Kajiyama, Shinya Toyokuni, Masaaki Mizuno, Masaru Hori

APPLIED PHYSICS EXPRESS Vol. 15 ( 5 ) 2022.5

Language：English Publishing type：Research paper (scientific journal) Publisher：IOP Publishing Ltd

Ringer's lactate solution was irradiated with non-equilibrium plasma under airtight conditions. The plasma-activated lactate (PAL) was produced with argon, oxygen, and nitrogen gases following purging of Ar. Cytotoxicity could be controlled by diluting PAL, and a killing effect was selectively obtained on cancer cells compared to normal cells for Ar+O-2+N-2 PALs. Nonetheless, cytotoxicity was partly reproduced by similar concentrations of H2O2 and NO2 (-) in the PALs. The organics produced by plasma irradiation to lactate were investigated using nuclear magnetic resonance, and the generation of methyl amino species was confirmed.

DOI： 10.35848/1882-0786/ac6360

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