研究者基本情報

• 博士 (獣医学), 北海道大学, 2020年03月

• https://researchmap.jp/bo-tomoki

• 90878141

• https://orcid.org/0000-0003-2395-2779

• 202001014650062881

• がん代謝
• 放射線腫瘍学
• 放射線生物学
• ミトコンドリア

• その他, その他, 放射線影響
• ライフサイエンス, 獣医学
• ライフサイエンス, 放射線科学
• ライフサイエンス, 腫瘍生物学
• 環境・農学, 放射線影響

• 学士課程, 獣医学部
• 博士課程, 獣医学院

研究活動情報

• 2025年05月, 日本酸化ストレス学会, 学術奨励賞
• 2023年09月, International Congress of Radiation Research 2023, Early Career Investigator Award in ICRR2023
  Heme biosynthesis is upregulated to enhance oxidative phosphorylation for cell survival after X-irradiation.
• 2021年05月, 第74回日本酸化ストレス学会/第21回日本NO学会 合同学術集会, 一般演題・ポスター優秀賞
• 2019年08月, European Radiation Research Society, Young Investigator Award 2019 in ICRR2019
• 2019年03月, 加藤記念バイオサイエンス振興財団, 加藤記念国際交流助成
• 2018年11月, 日本放射線影響学会第61回大会, 優秀演題発表賞
• 2017年10月, 日本放射線影響学会第60回大会, 優秀演題発表賞
• 2015年05月, Intenational Congress of Radiation Research, Young Investigator Award 2015 in ICRR2015
• 2015年05月, Intenational Congress of Radiation Research, Excellent Poster Award in ICRR2015

• Advances in redox imaging for cancer diagnosis and therapy
  Hironobu Yasui; Kazuhiro Kato; Tomoki Bo; Osamu Inanami
  Journal of Clinical Biochemistry and Nutrition, 78, 1, 14, 18, The Society for Free Radical Research Japan, 2026年, ［査読有り］
  研究論文（学術雑誌）
• Dephosphorylation of branched-chain α-keto acid dehydrogenase E1α (BCKDHA) promotes branched-chain amino acid catabolism and renders cancer cells resistant to X-rays by mitigating DNA damage.
  Tomoki Bo; Tsukasa Osaki; Junichi Fujii
  Biochemical and biophysical research communications, 742, 151154, 151154, 2025年01月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Branched-chain amino acids (BCAAs) facilitate cancer cell proliferation and survival. Stresses, including X-irradiation, increase BCAA uptake. However, the role of BCAA metabolism in cancer cell survival remains unclear. Therefore, this study aimed to elucidate the role of the BCAA catabolic pathway in cancer cell survival following X-irradiation. X-irradiation dose-dependently dephosphorylated branched-chain α-keto acid dehydrogenaseE1α (BCKDHA) suggesting the activation of the BCKDH complex, which catalyzes the rate-determining step of BCAA catabolism. We considered that activation of BCKDH promoted the BCAA catabolism, which resulted in cancer cell resistance to X-irradiation. Consistent with this notion, cells with BCKDHA knockdown exhibited increased radiosensitivity, which was associated with the increase in mitotic catastrophe and residual double-strand breaks by decreasing cellular ATP levels after X-irradiation. Our results suggest that BCKDHA dephosphorylation promotes BCAA catabolism, leading to cell survival by mitigating DNA damage after X-irradiation. Thus, BCAA catabolic pathway may be a target for radiation therapy.
• Primary Roles of Branched Chain Amino Acids (BCAAs) and Their Metabolism in Physiology and Metabolic Disorders.
  Tomoki Bo; Junichi Fujii
  Molecules (Basel, Switzerland), 30, 1, 2024年12月27日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Leucine, isoleucine, and valine are collectively known as branched chain amino acids (BCAAs) and are often discussed in the same physiological and pathological situations. The two consecutive initial reactions of BCAA catabolism are catalyzed by the common enzymes referred to as branched chain aminotransferase (BCAT) and branched chain α-keto acid dehydrogenase (BCKDH). BCAT transfers the amino group of BCAAs to 2-ketoglutarate, which results in corresponding branched chain 2-keto acids (BCKAs) and glutamate. BCKDH performs an oxidative decarboxylation of BCKAs, which produces their coenzyme A-conjugates and NADH. BCAT2 in skeletal muscle dominantly catalyzes the transamination of BCAAs. Low BCAT activity in the liver reduces the metabolization of BCAAs, but the abundant presence of BCKDH promotes the metabolism of muscle-derived BCKAs, which leads to the production of glucose and ketone bodies. While mutations in the genes responsible for BCAA catabolism are involved in rare inherited disorders, an aberrant regulation of their enzymatic activities is associated with major metabolic disorders such as diabetes, cardiovascular disease, and cancer. Therefore, an understanding of the regulatory process of metabolic enzymes, as well as the functions of the BCAAs and their metabolites, make a significant contribution to our health.
• Double-decker cage reduces mount frequency and ejaculation latency, resulting in reduced weight loss in male rats after mating behavior.
  Tomoki Bo; Naoki Fukuda; Junko Ozaki; Ayumi Inoue; Kiyoaki Katahira; Tunekata Ito
  Experimental animals, 2024年05月30日, ［査読有り］, ［筆頭著者, 責任著者］, ［国内誌］
  英語, 研究論文（学術雑誌）, Rats were the first mammals to be domesticated for scientific research, and abundant physiological data are available on them. Rats are expected to continue to play an important role as experimental animals, especially with advancements such as CRISPR/Cas9 technology. Environmental enrichment aims to promote species-specific behaviors and psychological well-being. In the present study, we designed a double-decker (DD) cage, which utilizes two stacked plastic cages for rat enrichment, and investigated the influence of housing in the DD cage on rat mating behavior. The results indicated that mount frequency, total mount counts, and total ejaculation latency were significantly lower in the DD cages than in the single-decker (SD) cages. Notably, in the DD cages, the body weight loss of male rats after mating behavior was lower than that observed in the SD cage. Water consumption per day during mating behavior was also significantly lower in the DD cages, although no significant differences were observed in daily food intake during mating behavior. In addition, reproductive performance, including pregnancy rate and birth rate, did not change in the DD cages. In summary, our study demonstrated that DD cages reduce mount frequency and ejaculation latency during rat mating, resulting in decreased water consumption and weight loss in male rats. Therefore, housing in DD cages may serve as a beneficial enrichment for rats.
• Mechanism of the Radioresistant Colorectal Cancer Cell Line SW480RR Established after Fractionated X Irradiation.
  Koya Yamashita; Hironobu Yasui; Tomoki Bo; Masaki Fujimoto; Osamu Inanami
  Radiation research, 2024年05月23日, ［査読有り］, ［国際誌］
  英語, 研究論文（学術雑誌）, Radioresistant cancer cells are risk factors for recurrence and are occasionally detected in recurrent tumors after radiotherapy. Intratumor heterogeneity is believed to be a potential cause of treatment resistance. Heterogeneity in DNA content has also been reported in human colorectal cancer; however, little is known about how such heterogeneity changes with radiotherapy or how it affects cancer radioresistance. In the present study, we established radioresistant clone SW480RR cells after fractionated X-ray irradiation of human colorectal cancer-derived SW480.hu cells, which are composed of two cell populations with different chromosome numbers, and examined how cellular radioresistance changed with fractionated radiotherapy. Compared with the parental cell population, which mostly comprised cells with higher ploidy, the radioresistant clones showed lower ploidy and less initial DNA damage. The lower ploidy cells in the parental cell population were identified as having radioresistance prior to irradiation; thus, SW480RR cells were considered intrinsically radioresistant cells selected from the parental population through fractionated irradiation. This study presents a practical example of the emergence of radioresistant cells from a cell population with ploidy heterogeneity after irradiation. The most likely mechanism is the selection of an intrinsically radioresistant population after fractionated X-ray irradiation, with a background in which lower ploidy cells exhibit lower initial DNA damage.
• Ascorbic Acid Protects Bone Marrow from Oxidative Stress and Transient Elevation of Corticosterone Caused by X-ray Exposure in Akr1a-Knockout Mice.
  Tomoki Bo; Hidekazu Nohara; Ken-Ichi Yamada; Satoshi Miyata; Junichi Fujii
  Antioxidants, 13, 2, 2024年01月25日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Bone marrow cells are the most sensitive to exposure to X-rays in the body and are selectively damaged even by doses that are generally considered permissive in other organs. Ascorbic acid (Asc) is a potent antioxidant that is reported to alleviate damages caused by X-ray exposure. However, rodents can synthesize Asc, which creates difficulties in rigorously assessing its effects in such laboratory animals. To address this issue, we employed mice with defects in their ability to synthesize Asc due to a genetic ablation of aldehyde reductase (Akr1a-KO). In this study, concentrations of white blood cells (WBCs) were decreased 3 days after exposure to X-rays at 2 Gy and then gradually recovered. At approximately one month, the recovery rate of WBCs was delayed in the Akr1a-KO mouse group, which was reversed via supplementation with Asc. Following exposure to X-rays, Asc levels decreased in plasma, bone marrow cells, and the liver during an early period, and then started to increase. X-ray exposure stimulated the pituitary gland to release adrenocorticotropic hormone (ACTH), which stimulated corticosterone secretion. Asc released from the liver, which was also stimulated by ACTH, appeared to be recruited to the bone marrow. Since corticosterone in high doses is injurious, these collective results imply that Asc protects bone marrow via its antioxidant capacity against ROS produced via exposure to X-rays and the cytotoxic action of transiently elevated corticosterone.
• Heme Biosynthesis is Crucial for Cell Survival and Mitochondrial OXPHOS after X Irradiation.
  Tomoki Bo; Koen Van Wijk; Osamu Nakajima
  Radiation research, 201, 1, 48, 54, 2024年01月01日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Heme is an essential component of the hemoproteins involved in the mitochondrial electron transport chain (ETC). Cancer cells have been reported to display high heme levels and increased activity of heme-containing proteins. Consistently, inhibition of heme biosynthesis by the ALAD inhibitor succinylacetone (SA) has been shown to reduce tumor cell survival. These observations indicate that heme biosynthesis is essential for cancer cell proliferation. X irradiation has been shown to increase mitochondrial mass, membrane potential, oxygen consumption, reactive oxygen species (ROS) production, and ATP synthesis. This finding suggests that radiation activates mitochondrial oxidative phosphorylation (OXPHOS). However, although heme is an essential component of the mitochondrial ETC, whether radiation influences heme biosynthesis remains unclear. In this study, we evaluated heme biosynthesis activity after X irradiation and examined the effects of heme biosynthesis inhibition by SA on cellular radiosensitivity and mitochondrial OXPHOS function. We demonstrated that X irradiation significantly increased ALAS1 mRNA levels and cellular heme content. Inhibition of heme biosynthesis by SA significantly decreased cellular heme content and sensitized cancer cells to radiation. We also showed that SA reduced cellular ATP levels, mitochondrial membrane potential, and mitochondrial ROS production, suggesting mitochondrial OXPHOS dysfunction. SA decreased the expression of mitochondrial heme-related proteins COX2 and cytochrome c but did not influence COX1 and VDAC expression. These results indicate that inhibition of heme biosynthesis decreased mitochondrial ETC protein expression and OXPHOS activity, which triggered cellular ATP depletion and radiosensitization after X irradiation. In summary, heme biosynthesis is upregulated by X irradiation and is essential for mitochondrial OXPHOS and cell survival.
• Ascorbate Is a Primary Antioxidant in Mammals
  Junichi Fujii; Tsukasa Osaki; Tomoki Bo
  MOLECULES, 27, 19, 2022年10月, ［査読有り］
  英語
• Metformin preferentially enhances the radio-sensitivity of cancer stem-like cells with highly mitochondrial respiration ability in HMPOS.
  Tatsuya Deguchi; Kenji Hosoya; Shango Kim; Yusuke Murase; Kumiko Yamamoto; Tomoki Bo; Hironobu Yasui; Osamu Inanami; Mahiro Okumura
  Molecular therapy oncolytics, 22, 143, 151, 2021年09月24日, ［査読有り］, ［国際誌］
  英語, 研究論文（学術雑誌）, Metformin has many anti-cancer effects, alone or in combination with radiation. However, the mechanism underlying its radio-sensitized effect is still unclear, especially for cancer stem-like cells (CSCs). Here, the radio-sensitized effect of metformin was investigated, and its mechanism was revealed in CSCs derived from canine osteosarcoma cell line (HMPOS), a canine osteosarcoma cell line. Spheroid cells (SCs) were used as CSCs-rich cells derived from sphere formation, and SCs were compared with normal adherent culture cells (ACs). The radio-sensitizing effect of metformin using clonogenic assay and tumor growth in mice xenograft model were evaluated, and the mechanism of its radio-sensitization focusing on mitochondrial function was revealed. Metformin significantly enhanced radio-sensitivity of SCs through its inhibition of the mitochondrial function, as shown by decreased oxygen consumption, decreased mitochondrial membrane potential, and decreased ATP production. Additionally, SCs had a higher ability of mitochondrial respiration than ACs, which may have caused difference of their sensitivity of metformin and irradiation. In conclusion, mitochondrial function might play an important role in the sensitivity of metformin and irradiation, and drugs that target mitochondrial respiration, such as metformin, are promising radio-sensitizers to target CSCs.
• Eribulin improves tumor oxygenation demonstrated by 18F-DiFA hypoxia imaging, leading to radio-sensitization in human cancer xenograft models.
  Tomoki Bo; Hironobu Yasui; Tohru Shiga; Yuki Shibata; Masaki Fujimoto; Motofumi Suzuki; Kei Higashikawa; Naoki Miyamoto; Osamu Inanami; Yuji Kuge
  European journal of nuclear medicine and molecular imaging, 2021年09月01日, ［査読有り］, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, PURPOSE: Eribulin, an inhibitor of microtubule dynamics, is known to show antitumor effects through its remodeling activity in the tumor vasculature. However, the extent to which the improvement of tumor hypoxia by eribulin affects radio-sensitivity remains unclear. We utilized 1-(2,2-dihydroxymethyl-3-18F-fluoropropyl)-2-nitroimidazole (18F-DiFA), a new PET probe for hypoxia, to investigate the effects of eribulin on tumor hypoxia and evaluate the radio-sensitivity during eribulin treatment. METHODS: Mice bearing human breast cancer MDA-MB-231 cells or human lung cancer NCI-H1975 cells were administered a single dose of eribulin. After administration, mice were injected with 18F-DiFA and pimonidazole, and tumor hypoxia regions were analyzed. For the group that received combined treatment with radiation, 18F-DiFA PET/CT imaging was performed before tumors were locally X-irradiated. Tumor size was measured every other day after irradiation. RESULTS: Eribulin significantly reduced 18F-DiFA accumulation levels in a dose-dependent manner. Furthermore, the reduction in 18F-DiFA accumulation levels by eribulin was most significant 7 days after treatment. These results were also supported by reduction of the pimonidazole-positive hypoxic region. The combined treatment showed significant retardation of tumor growth in comparison with the control, radiation-alone, and drug-alone groups. Importantly, tumor growth after irradiation was inversely correlated with 18F-DiFA accumulation. CONCLUSION: These results demonstrated that 18F-DiFA PET/CT clearly detected eribulin-induced tumor oxygenation and that eribulin efficiently enhanced the antitumor activity of radiation by improving tumor oxygenation.
• LAT1 inhibitor JPH203 sensitizes cancer cells to radiation by enhancing radiation-induced cellular senescence.
  Tomoki Bo; Sho Kobayashi; Osamu Inanami; Junichi Fujii; Osamu Nakajima; Tsunekata Ito; Hironobu Yasui
  Translational oncology, 14, 11, 101212, 101212, 2021年08月27日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, L-type amino acid transporter 1 (LAT1) is important for transporting neutral amino acids into cells. LAT1 expression is correlated with cancer malignancy, suggesting that LAT1 is a promising target for cancer therapy. JPH203, a potential novel drug targeting LAT1, has been shown to suppress tumor growth in various cancer cell lines. However, a combination study of JPH203 and radiation therapy has not been reported. Here, we examined the effects of JPH203 on radiosensitivity after irradiation in A549 and MIA Paca-2 cells. We showed that X-irradiation increased cellular neutral amino acid uptake via LAT1 in both cell lines. JPH203 inhibited the radiation-induced increase in neutral amino acid uptake. We demonstrated that JPH203, at minimally toxic concentrations, significantly sensitized cancer cells to radiation. JPH203 significantly downregulated mTOR activity and enhanced cellular senescence post-irradiation without reducing ATP and GSH levels. These results indicate that LAT1 inhibition by JPH203 sensitizes cancer cells to radiation by enhancing cellular senescence via mTOR downregulation. Thus, JPH203 may be a potent anti-cancer drug in combination with radiation therapy.
• Radiation-induced abnormal centrosome amplification and mitotic catastrophe in human cervical tumor HeLa cells and murine mammary tumor EMT6 cells.
  Masaki Fujimoto; Tomoki Bo; Kumiko Yamamoto; Hironobu Yasui; Tohru Yamamori; Osamu Inanami
  Journal of clinical biochemistry and nutrition, 67, 3, 240, 247, 2020年11月, ［査読有り］, ［国内誌］
  英語, 研究論文（学術雑誌）, Mitotic catastrophe is a form of cell death linked to aberrant mitosis caused by improper or uncoordinated mitotic progression. Abnormal centrosome amplification and mitotic catastrophe occur simultaneously, and some cells with amplified centrosomes enter aberrant mitosis, but it is not clear whether abnormal centrosome amplification triggers mitotic catastrophe. Here, to investigate whether radiation-induced abnormal centrosome amplification is essential for induction of radiation-induced mitotic catastrophe, centrinone-B, a highly selective inhibitor of polo-like kinase 4, was utilized to inhibit centrosome amplification, since polo-like kinase 4 is an essential kinase in centrosome duplication. When human cervical tumor HeLa cells and murine mammary tumor EMT6 cells were irradiated with 2.5 Gy of X-rays, cells with morphological features of mitotic catastrophe and the number of cells having >2 centrosomes increased in both cell lines. Although centrinone-B significantly inhibited radiation-induced abnormal centrosome amplification in both cell lines, such treatment did not change cell growth and significantly enhanced mitotic catastrophe in HeLa cells exposed to X-rays. In contrast, inhibition of centrosome amplification reduced cell growth and mitotic catastrophe in EMT6 cells exposed to X-rays. These results indicated that the role of radiation-induced abnormal centrosome amplification in radiation-induced mitotic catastrophe changes, depending on the cell type.
• Mitochondrial fission promotes radiation-induced increase in intracellular Ca2+ level leading to mitotic catastrophe in mouse breast cancer EMT6 cells
  Tomoki Bo; Tohru Yamamori; Kumiko Yamamoto; Masaki Fujimoto; Hironobu Yasui; Osamu Inanami
  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 522, 1, 144, 150, 2020年01月, ［査読有り］, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）
• Genotoxic Responses of Mitochondrial Oxygen Consumption Rate and Mitochondrial Semiquinone Radicals in Tumor Cells
  Kumiko Yamamoto; Hironobu Yasui; Tomoki Bo; Tohru Yamamori; Wakako Hiraoka; Toshihide Yamasaki; Ken-ichi Yamada; Osamu Inanami
  APPLIED MAGNETIC RESONANCE, 49, 8, 837, 851, 2018年08月, ［査読有り］
  英語, 研究論文（学術雑誌）
• Calmodulin-dependent protein kinase II (CaMKII) mediates radiation-induced mitochondrial fission by regulating the phosphorylation of dynamin-related protein 1 (Drp1) at serine 616.
  Tomoki Bo; Tohru Yamamori; Motofumi Suzuki; Yuri Sakai; Kumiko Yamamoto; Osamu Inanami
  Biochemical and biophysical research communications, 495, 2, 1601, 1607, 2018年01月08日, ［査読有り］, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Mitochondrial dynamics are suggested to be indispensable for the maintenance of cellular quality and function in response to various stresses. While ionizing radiation (IR) stimulates mitochondrial fission, which is mediated by the mitochondrial fission protein, dynamin-related protein 1 (Drp1), it remains unclear how IR promotes Drp1 activation and subsequent mitochondrial fission. Therefore, we conducted this study to investigate these concerns. First, we found that X-irradiation triggered Drp1 phosphorylation at serine 616 (S616) but not at serine 637 (S637). Reconstitution analysis revealed that introduction of wild-type (WT) Drp1 recovered radiation-induced mitochondrial fission, which was absent in Drp1-deficient cells. Compared with cells transfected with WT or S637A Drp1, the change in mitochondrial shape following irradiation was mitigated in S616A Drp1-transfected cells. Furthermore, inhibition of CaMKII significantly suppressed Drp1 S616 phosphorylation and mitochondrial fission induced by IR. These results suggest that Drp1 phosphorylation at S616, but not at S637, is prerequisite for radiation-induced mitochondrial fission and that CaMKII regulates Drp1 phosphorylation at S616 following irradiation.
• NADPH oxidase 4 mediates ROS production in radiation-induced senescent cells and promotes migration of inflammatory cells.
  Yuri Sakai; Tohru Yamamori; Yoji Yoshikawa; Tomoki Bo; Motofumi Suzuki; Kumiko Yamamoto; Tetsuro Ago; Osamu Inanami
  Free radical research, 52, 1, 92, 102, 2018年01月, ［査読有り］, ［国際誌］
  英語, 研究論文（学術雑誌）, Excessive DNA damage induced by ionising radiation (IR) to normal tissue cells is known to trigger cellular senescence, a process termed stress-induced premature senescence (SIPS). SIPS is often accompanied by the production of reactive oxygen species (ROS), and this is reported to be important for the initiation and maintenance of SIPS. However, the source of ROS during SIPS after IR and their significance in radiation-induced normal tissue damage remain elusive. In the present study, we tested the hypothesis that the NADPH oxidase (NOX) family of proteins mediates ROS production in SIPS-induced cells after IR and plays a role in SIPS-associated biological events. X-irradiation of primary mouse embryonic fibroblasts (MEFs) resulted in cellular senescence and the concomitant increase of intracellular ROS. Among all six murine NOX isoforms (NOX1-4 and DUOX1/2), only NOX4 was detectable under basal conditions and was upregulated following IR. In addition, radiation-induced ROS production was diminished by genetic or pharmacological inhibition of NOX4. Meanwhile, NOX4 deficiency did not affect the induction of cellular senescence after IR. Furthermore, the migration of human monocytic U937 cells to the culture medium collected from irradiated MEFs was significantly reduced by NOX4 inhibition, suggesting that NOX4 promotes the recruitment of inflammatory cells. Collectively, our findings imply that NOX4 mediates ROS production in radiation-induced senescent cells and contributes to normal tissue damage after IR via the recruitment of inflammatory cells and the exacerbation of tissue inflammation.
• Evaluation of mitochondrial redox status and energy metabolism of X-irradiated HeLa cells by LC/UV, LC/MS/MS and ESR
  Kumiko Yamamoto; Yoshinori Ikenaka; Takahiro Ichise; Tomoki Bo; Mayumi Ishizuka; Hironobu Yasui; Wakako Hiraoka; Tohru Yamamori; Osamu Inanami
  FREE RADICAL RESEARCH, 52, 6, 648, 660, 2018年, ［査読有り］, ［国際誌］
  英語, 研究論文（学術雑誌）
• MK-8776, a novel Chk1 inhibitor, exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis.
  Motofumi Suzuki; Tohru Yamamori; Tomoki Bo; Yuri Sakai; Osamu Inanami
  Translational oncology, 10, 4, 491, 500, 2017年08月, ［査読有り］, ［国際誌］
  英語, Checkpoint kinase 1 (Chk1) is an evolutionarily conserved serine/threonine kinase that plays an important role in G2/M checkpoint signaling. Here, we evaluate the radiosensitizing effects of a novel selective Chk1 inhibitor MK-8776, comparing its efficacy with a first-generation Chk1 inhibitor UCN-01, and attempt to elucidate the mechanism of radiosensitization. In a clonogenic survival assay, MK-8776 demonstrated a more pronounced radiosensitizing effect than UCN-01, with lower cytotoxicity. Importantly, radiosensitization by MK-8776 can be achieved at doses as low as 2.5 Gy, which is a clinically applicable irradiation dose. MK-8776, but not UCN-01, exacerbated mitotic catastrophe (MC) and centrosome abnormalities, without affecting repair kinetics of DNA double strand breaks. Furthermore, live-cell imaging revealed that MK-8776 significantly abrogated the radiation-induced G2/M checkpoint, prolonged the mitotic phase, and enhanced aberrant mitosis. This suggests that Chk1 inhibition by MK-8776 activates a spindle assembly checkpoint and increases mitotic defects in irradiated EMT6 cells. In conclusion, we have shown that, at minimally toxic concentrations, MK-8776 enhances radiation-induced cell death through the enhancement of aberrant mitosis and MC, without affecting DNA damage repair.
• Analysis of the mechanism of radiation-induced upregulation of mitochondrial abundance in mouse fibroblasts.
  Tohru Yamamori; Tomoya Sasagawa; Osamu Ichii; Mie Hiyoshi; Tomoki Bo; Hironobu Yasui; Yasuhiro Kon; Osamu Inanami
  Journal of radiation research, 58, 3, 292, 301, 2017年05月01日, ［査読有り］, ［国際誌］
  英語, 研究論文（学術雑誌）, Mitochondria strongly contribute to the maintenance of cellular integrity through various mechanisms, including oxidative adenosine triphosphate production and calcium homeostasis regulation. Therefore, proper regulation of the abundance, distribution and activity of mitochondria is crucial for the maintenance of cellular homeostasis. Previous studies have shown that ionizing radiation (IR) alters mitochondrial functions, suggesting that mitochondria are likely to be an important target of IR. Though IR reportedly influences cellular mitochondrial abundance, the mechanism remains largely unknown. In this study, we examined how IR influences mitochondrial abundance in mouse fibroblasts. When mouse NIH/3T3 cells were exposed to X-rays, a time-dependent increase was observed in mitochondrial DNA (mtDNA) and mitochondrial mass, indicating radiation-induced upregulation of mitochondrial abundance. Meanwhile, not only did we not observe a significant change in autophagic activity after irradiation, but in addition, IR hardly influenced the expression of two mitochondrial proteins, cytochrome c oxidase subunit IV and cytochrome c, or the mRNA expression of Polg, a component of DNA polymerase γ. We also observed that the expression of transcription factors involved in mitochondrial biogenesis was only marginally affected by IR. These data imply that radiation-induced upregulation of mitochondrial abundance is an event independent of macroautophagy and mitochondrial biogenesis. Furthermore, we found evidence that IR induced long-term cell cycle arrest and cellular senescence, indicating that these events are involved in regulating mitochondrial abundance. Considering the growing significance of mitochondria in cellular radioresponses, we believe the present study provides novel insights into understanding the effects of IR on mitochondria.
• Lipophilic triphenylphosphonium derivatives enhance radiation-induced cell killing via inhibition of mitochondrial energy metabolism in tumor cells.
  Hironobu Yasui; Kumiko Yamamoto; Motofumi Suzuki; Yuri Sakai; Tomoki Bo; Masaki Nagane; Eri Nishimura; Tohru Yamamori; Toshihide Yamasaki; Ken-Ichi Yamada; Osamu Inanami
  Cancer letters, 390, 160, 167, 2017年04月01日, ［査読有り］, ［国際誌］
  英語, 研究論文（学術雑誌）, It has recently been reported that radiation enhances mitochondrial energy metabolism in various tumor cell lines. To examine how this radiation-induced alteration in mitochondrial function influences tumor cell viability, various lipophilic triphenylphosphonium (TPP+) cation derivatives and related compounds such as 4-hydroxy-2,2,6,6-tetramethyl-1-oxy-piperidin (Tempol) with TPP+ (named "Mito-") were designed to inhibit the mitochondrial electron transport chain. Mito-(CH2)10-Tempol (M10T) and its derivatives, Mito-(CH2)5-Tempol (M5T), Mito-(CH2)10-Tempol-Methyl (M10T-Me), Mito-C10H21 (M10), and C10H21-Tempol (10T), were prepared. In HeLa human cervical adenocarcinoma cells and A549 human lung carcinoma cells, the fractional uptake of the compound into mitochondria was highest among the TTP+ analogs conjugated with Tempol (M10T, M5T, and 10T). M10T, M10T-Me, and M10 exhibited strong cytotoxicity and enhanced X-irradiation-induced reproductive cell death, while 10T and M5T did not. Furthermore, M10T, M10T-Me, and M10 decreased basal mitochondrial membrane potential and intracellular ATP. M10T treatment inhibited X-ray-induced increases in ATP production. These results indicate that the TPP cation and a long hydrocarbon linker are essential for radiosensitization of tumor cells. The reduction in intracellular ATP by lipophilic TPP+ is partly responsible for the observed radiosensitization.
• Inhibition of the mitochondrial fission protein dynamin-related protein 1 (Drp1) impairs mitochondrial fission and mitotic catastrophe after x-irradiation.
  Tohru Yamamori; Satoshi Ike; Tomoki Bo; Tomoya Sasagawa; Yuri Sakai; Motofumi Suzuki; Kumiko Yamamoto; Masaki Nagane; Hironobu Yasui; Osamu Inanami
  Molecular biology of the cell, 26, 25, 4607, 17, 2015年12月15日, ［査読有り］, ［国際誌］
  英語, 研究論文（学術雑誌）, Accumulating evidence suggests that mitochondrial dynamics is crucial for the maintenance of cellular quality control and function in response to various stresses. However, the role of mitochondrial dynamics in cellular responses to ionizing radiation (IR) is still largely unknown. In this study, we provide evidence that IR triggers mitochondrial fission mediated by the mitochondrial fission protein dynamin-related protein 1 (Drp1). We also show IR-induced mitotic catastrophe (MC), which is a type of cell death associated with defective mitosis, and aberrant centrosome amplification in mouse embryonic fibroblasts (MEFs). These are attenuated by genetic or pharmacological inhibition of Drp1. Whereas radiation-induced aberrant centrosome amplification and MC are suppressed by the inhibition of Plk1 and CDK2 in wild-type MEFs, the inhibition of these kinases is ineffective in Drp1-deficient MEFs. Furthermore, the cyclin B1 level after irradiation is significantly higher throughout the time course in Drp1-deficient MEFs than in wild-type MEFs, implying that Drp1 is involved in the regulation of cyclin B1 level. These findings strongly suggest that Drp1 plays an important role in determining the fate of cells after irradiation via the regulation of mitochondrial dynamics.

• DNA量の不均一性を有するがん細胞株における放射線分割照射後の放射線抵抗性がん細胞の出現機構の解析
  山下晃矢; 安井博宣; 房知輝; 藤本政毅; 房知輝; 藤本政毅; 稲波修, 日本獣医学会学術集会講演要旨集, 167th, 2024年
• 放射線照射後のミトコンドリア形態変化が感受性を調節する
  房知輝; 房知輝; 山盛徹; 安井博宣; 稲波修, 放射線生物研究, 55, 4, 2020年, ［査読有り］

• 種々のがん細胞におけるミトコンドリア電子伝達系の放射線応答の解析
  山本 久美子; 安井 博宣; 藤本 政毅; 房 知輝; 稲波 修
  日本獣医学会学術集会講演要旨集, 2019年08月, (公社)日本獣医学会, 日本語, その他
  2019年08月 - 2019年08月, ［国際会議］
• ミトコンドリア分裂はCa2+制御を通じて放射線による分裂期崩壊誘導に寄与する
  房 知輝; 山盛 徹; 山本 久美子; 藤本 政毅; 安井 博宣; 稲波 修
  日本獣医学会学術集会講演要旨集, 2019年08月, (公社)日本獣医学会, 日本語, その他
  2019年08月 - 2019年08月, ［国際会議］
• ヒト肺腺がん由来A549細胞におけるグルタミノリシスを標的とする放射線増感効果
  藤本 政毅; 房 知輝; 山本 久美子; 安井 博宣; 稲波 修
  日本獣医学会学術集会講演要旨集, 2019年08月, (公社)日本獣医学会, 日本語, その他
  2019年08月 - 2019年08月, ［国際会議］
• ヒト肺がん由来A549細胞における解糖系の放射線応答性に関する研究
  福島 佑一郎; 安井 博宜; 藤本 政毅; 山本 久美子; 房 知輝; 稲波 修
  日本獣医学会学術集会講演要旨集, 2019年08月, (公社)日本獣医学会, 日本語, その他
  2019年08月 - 2019年08月, ［国際会議］
• Glutamine代謝阻害がX線照射による早期細胞老化およびアポトーシスの誘導に与える影響の検討
  東山 りつ子; 安井 博宣; 房 知輝; 山本 久美子; 藤本 政毅; 稲波 修
  日本獣医学会学術集会講演要旨集, 2019年08月, (公社)日本獣医学会, 日本語, その他
  2019年08月 - 2019年08月, ［国際会議］
• 放射線により引き起こされる分裂期崩壊に対するミトコンドリア分裂の寄与メカニズムの解析
  房 知輝; 山盛 徹; 山本 久美子; 稲波 修
  日本放射線影響学会大会講演要旨集, 2018年11月, (一社)日本放射線影響学会, 日本語, その他
  2018年11月 - 2018年11月, ［国際会議］
• ESR法による新規ミトコンドリア機能評価法を用いたがん細胞の放射線応答の解析
  山本 久美子; 安井 博宣; 房 知輝; 山盛 徹; 稲波 修
  日本放射線影響学会大会講演要旨集, 2018年11月, (一社)日本放射線影響学会, 日本語, その他
  2018年11月 - 2018年11月, ［国際会議］
• Centrinonne-Bによる中心体複製阻害が放射線誘発分裂期崩壊に与える影響
  藤本 政毅; 山盛 徹; 房 知輝; 山本 久美子; 稲波 修
  日本放射線影響学会大会講演要旨集, 2018年11月, (一社)日本放射線影響学会, 日本語, その他
  2018年11月 - 2018年11月, ［国際会議］
• 新規ミトコンドリア呼吸機能評価法を用いたがん細胞に対する放射線影響の評価
  山本 久美子; 安井 博宣; 房 知輝; 山盛 徹; 稲波 修
  日本獣医学会学術集会講演要旨集, 2018年08月, (公社)日本獣医学会, 日本語, その他
  2018年08月 - 2018年08月, ［国際会議］
• ミトコンドリア分裂抑制が細胞の放射線感受性低下を引き起こすメカニズムの解析
  房 知輝; 山盛 徹; 山本 久美子; 稲波 修
  日本獣医学会学術集会講演要旨集, 2018年08月, (公社)日本獣医学会, 日本語, その他
  2018年08月 - 2018年08月, ［国際会議］

• 2019年06月 - 現在
  日本酸化ストレス学会
• 2018年04月 - 現在
  日本獣医学会
• 2015年04月 - 現在
  日本放射線影響学会

• がん窒素代謝を標的とした新規放射線療法開発のための基盤研究
  科学研究費助成事業
  2024年04月01日 - 2027年03月31日
  房 知輝
  日本学術振興会, 若手研究, 山形大学, 24K18014
• ポリアミン-アクロレイン経路の亢進による活性化マクロファージの制御機構
  科学研究費助成事業
  2024年04月 - 2027年03月
  藤井 順逸; 房 知輝
  日本学術振興会, 基盤研究(C), 山形大学, 24K10074
• 放射線による中性アミノ酸代謝動態の解析とこれを標的とした新規がん治療法の開発
  科学研究費助成事業 若手研究
  2021年04月01日 - 2024年03月31日
  房 知輝
  がん細胞では糖代謝に加えてアミノ酸代謝にもリプログラミングが生じており、特定のアミノ酸に代謝を依存するがん細胞種も複数報告されている。その中で、中性アミノ酸ががんの生存に対して果たす役割をエネルギー代謝の観点から報告した知見はこれまでにない。申請者は、がん細胞内の中性アミノ酸量が放射線により増加する結果を得たことから、放射線照射が中性アミノ酸代謝を亢進し、これが放射線照射後の細胞生存に働くのではないかと仮説を立てた。本研究では仮説立証のために、がん細胞における中性アミノ酸の動態を エネルギー代謝の面から解析し、中性アミノ酸代謝の放射線応答についても調べる。これらの検討から、がん細胞の中性アミノ酸代謝を標的とした新たながん治療法の開発を目指す。
  本年度はLC/MSを用いて解糖系ならびにTCA回路における代謝中間産物の測定法を確立した。また、
  分岐鎖アミノ酸の代謝変換を担う酵素の阻害剤またはRNAi処理がこれらの代謝中間産物に与える影響を明らかにした。加えて、コロニー形成法による放射線感受性の評価を実施したところ、上記の阻害剤が軽微ではあるが放射線増感効果を示すことを明らかにした。
  日本学術振興会, 若手研究, 山形大学, 21K14971
• 放射線照射時のアンモニア代謝動態の解析とそれを標的とした新規がん放射線療法の開発に向けた基礎的研究
  医学系研究助成
  2023年11月
  武田科学振興財団
• 中性アミノ酸輸送体LAT1を標的とした新規がん放射線療法の開発へ向けた基礎的研究
  科学研究費助成事業 研究活動スタート支援
  2020年09月11日 - 2022年03月31日
  房 知輝
  申請者は放射線が生存したがん細胞のエネルギー代謝を亢進することに加え、中性アミノ酸量を増加させることを明らかにしていたことから、中性アミノ酸代謝を標的としたがん放射線療法の可能ではないかと推測した。そこで、本研究では中性アミノ酸の細胞内への取込みに働くL-type amino acid transporter 1 (LAT1)の特異的阻害剤JPH203が放射線増感効果を引き起こせるかどうか検討した。その結果、JPH203が複数のがん細胞株で放射線感受性を増強した。また、その増感メカニズムにはmTORシグナル減弱を介した細胞老化の誘導が寄与すること明らかにした。
  日本学術振興会, 研究活動スタート支援, 山形大学, 20K22830
• 放射線によるミトコンドリア形態機能応答とそれらを標的とした新規がん治療法開発
  科学研究費助成事業 特別研究員奨励費
  2019年04月25日 - 2021年03月31日
  房 知輝
  ミトコンドリアの形態変化を通じて制御される機能変化が放射線感受性を修飾しているのではないかと考え、複数のミトコンドリア形態制御因子に着目し、これが細胞の放射線感受性に与える影響ならびに、その感受性変化メカニズムを明らかにすることを目的にin vitroレベルでの検討を行った。ミトコンドリア形態制御因子を安定的にノックダウンしたマウス乳がん由来EMT6細胞を作出し、放射線感受性を評価したところ、ミトコンドリア分裂制御因子であるDrp1およびFis1 KD細胞では放射線感受性が有意に低下していた一方で、ミトコンドリア融合制御因子であるMfn2およびOpa1 KD細胞では感受性に変化はなかった。このことから、放射線による細胞死にはミトコンドリア分裂が重要であることが示唆された。そこで、Drp1およびFis1 KD細胞で観察された感受性の変化を引き起こすメカニズムを明らかにすることを試みた。その結果、Drp1およびFis1 KDは分裂期崩壊による細胞死を一部抑制した。さらに、Ca2+キレート剤のBAPTA-AM処理による放射線照射後の分裂期崩壊を低減したことから、Ca2+が分裂期崩壊誘導に寄与していることが示唆された。さらに、X線照射が細胞内[Ca2+]iに与える影響を評価したところ、放射線が細胞内[Ca2+]iを増加させる一方で、ミトコンドリア分裂の抑制がこの増加を抑制することが明らかとなった。まとめると、放射線照射後にミトコンドリア分裂が亢進することで細胞内[Ca2+]iが増加し、これが分裂期崩壊の誘導に寄与することが示唆された。本年度の研究実施によりin vitroでのミトコンドリア形態変化を介した細胞死を修飾するミトコンドリア機能変化を明らかにすることができた。
  日本学術振興会, 特別研究員奨励費, 北海道大学, 19J13470