Our Research 2017
(77) Analysis of damage in mouse liver from radon, X-ray, or alcohol treatments using a self-organizing map
March 28, 2017
Norie KANZAKI
(Graduate School of Health Sciences, Okayama University)
In our previous studies, we found that low-dose radiation inhibits oxidative stress induced diseases due to increased antioxidants. However, the analysis of oxidative stress is challenging, especially that of low levels of oxidative stress, because antioxidative substances are intricately involved. There are no reports comparing the different effects of oxidative stress caused by the various factors. The objective of this study was to compare radiation-induced liver damage with alcohol-induced that.
Mice were treated with radon inhalation, X-irradiation, or alcohol administration. We evaluated the oxidative damage levels in livers from the Self-organizing map (SOM) results for hepatic function and antioxidative substances. Here, we used the SOM for the analysis because it is a useful tool for the visualization of oxidative damage induced by different sources (Fig. 1).
The results showed that the effects of low-dose irradiation (radon inhalation at a concentration of up to 2000 Bq/m3, or X-irradiation at a dose of up to 2.0 Gy) were comparable with that of alcohol administration at 0.5 g/kg bodyweight. Analysis using the SOM to discriminate small changes was made possible by its ability to learn to adapt to unexpected changes. Moreover, when using a spherical SOM, the method comprehensively examined liver damage by radon, X-ray, or alcohol. We found that the types of liver damage caused by radon, X-rays, or alcohol have different characteristics.
This novel method using the SOM was successful in comprehensively examining oxidative liver damage, and our approach was useful for evaluating the oxidative stress caused by various life environmental factors. The results in this study presented provide a substantial basis for future studies aimed at elucidating the mechanisms involved in oxidative stress.
Fig1. Our method for evaluation of damage with antioxidants and functions in liver using SOM.
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日本語解説 「機械学習（自己組織化マップ）を用いたラドン・X線・アルコールによるマウス肝障害の比較」
東電福島第一原発事故以降，放射線の健康影響への不安対策は急務であり，本研究では，その健康不安払拭のため，より生活に密着したアルコールと放射線による肝臓への影響を比較評価した。すなわち，ラドン・X線・アルコール各々に曝露したマウス肝臓の機能と抗酸化機能を分析し，自己組織化マップ（SOM）を用い視覚的な影響評価をした。その結果，2000Bq/m3以下ラドン24時間吸入や2Gy以下X線全身照射の影響は0.5g/kg体重アルコール投与に相当すること，それらの酸化ストレスの特性に差があることがわかった。これより，本実験条件下での低線量放射線被曝は極微量の酸化ストレスであると推測できた。以上の所見より，遺伝子解析や肝疾患自動診断等に応用されるSOMは，抗酸化機能に着目した肝障害の評価にも有効であることがわかった。今後も，低線量放射線による酸化ストレスに関する新たな知識抽出・機構解明に機械学習を活用していきたい。
(78) Involvement of reactive oxygen species in ionizing radiation–induced upregulation of cell surface Toll-like receptor 2 and 4 expression in human monocytic cells
April 20, 2017
Hironori YOSHINO
(Hirosaki University Graduate School of Health Sciences)
Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogen-associated molecular patterns and are indispensable for antibacterial and antiviral immunity. Our previous report showed that ionizing radiation increases the cell surface expressions of TLR2 and TLR4 and enhances their responses to agonists in human monocytic THP1 cells [Yoshino et al., J. Rad. Res., 55:876-84, 2014]. The present study investigated how ionizing radiation increases the cell surface expressions of TLR2 and TLR4 in THP1 cells. The THP1 cells treated or not treated with pharmaceutical agents such as cycloheximide and N-acetyl-L-cysteine (NAC) were exposed to X-ray irradiation, following which the expressions of TLRs and mitogen-activated protein kinase were analyzed. X-ray irradiation increased the mRNA expressions of TLR2 and TLR4, and treatment with a protein synthesis inhibitor cycloheximide abolished the radiation-induced upregulation of their cell surface expressions. These results indicate that radiation increased those receptors through de novo protein synthesis. Furthermore, treatment with an antioxidant NAC suppressed not only the radiation-induced upregulation of cell surface expressions of TLR2 and TLR4, but also the radiation-induced activation of the c-Jun N-terminal kinase (JNK) pathway. Since it has been shown that the inhibitor for JNK can suppress the radiation-induced upregulation of TLR expression, the present results suggest that ionizing radiation increased the cell surface expressions of TLR2 and TLR4 through reactive oxygen species-mediated JNK activation.
(80) PU-H71, a novel Hsp90 inhibitor, as a potential cancer-specific sensitizer to carbon-ion beam therapy
June 15, 2017
Huizi Keiko Li
(Research Fellow of Japan Society for the Promotion of Science /
Graduate School of Medical and Pharmaceutical Sciences, Chiba University)
PU-H71, a heat shock protein 90 (Hsp90) inhibitor, has yielded therapeutic efficacy in many preclinical models and is currently in clinical trials. Carbon-ion radiotherapy (CIRT) has provided successful tumor control; however, there is still room for improvement, particularly in terms of tumor-specific radiosensitization. The Hsp90 inhibitor PU-H71 has been shown to sensitize tumor cells to X-ray radiation. A murine osteosarcoma cell line (LM8) and a normal human fibroblast cell line (AG01522) were treated with PU-H71 before X-ray, 14- or 50-keV/μm carbon-ion beam (C-ion) irradiation. Cell survival and protein expression were evaluated with colony formation and western blot, respectively. Treatment with PU-H71 alone was shown to be non-toxic to both cell lines; however, PU-H71 was shown to significantly sensitize LM8 cells to not only X-ray, but also to C-ion irradiation, while only a minimal sensitizing effect was observed in AG01522 cells. PU-H71 treatment was found to suppress the protein expression levels of Rad51 and Ku70, which are associated with the homologous recombination pathway and the non-homologous end-joining pathway of double-strand break repair. The findings reported here suggest that PU-H71 could be a promising radiosensitizer for CIRT.
(81) Locoregional therapy with α-emitting trastuzumab against peritoneal metastasis of human epidermal growth factor receptor 2-positive gastric cancer in mice
July 28, 2017
Huizi Keiko Li
(Research Fellow of Japan Society for the Promotion of Science /
Graduate School of Medical and Pharmaceutical Sciences, Chiba University)
Peritoneal metastasis of gastric cancer (PMGC) is incurable and thus has an extremely poor prognosis. We have found however that locoregionally administered trastuzumab armed with a radionuclide astatine-211 (211 At)-emitting α-particle (211At-trastuzumab) is effective against HER2-positive PMGC in a xenograft mouse model. We first observed that 211At-trastuzumab can specifically bind and effectively kill NCI-N87 (N87) cells, which are HER2-positive human metastatic GC cells, both in vitro and in subcutaneous tumors. We established a PMGC mouse model using N87 xenografts stably expressing luciferase to test α-particle radioimmunotherapy with 211At-trastuzumab against PMGC. Biodistribution analysis in this PMGC mouse model revealed that the intraperitoneal administration of 211At-trastuzumab (1 MBq) was a more efficient means of delivery of 211At into metastatic tumors than intravenous injection; the maximum tumor uptake with intraperitoneal administration was over 60 percent injected dose per gram (%ID/g) compared to about 18%ID/g with intravenous injection. Surprisingly, a single intraperitoneal injection of 211At-trastuzumab (1 MBq) was sufficient to completely eradicate intraperitoneally disseminated HER2-positive GC xenografts in two of six treated mice by inducing DNA double-strand breaks, and to drastically reduce the tumor burden in three further mice. No body weight loss, leukocytopenia or significant biochemical changes in liver and kidney function were observed in the treatment group. Accordingly, locoregionally administered 211At-trastuzumab significantly prolonged the survival time of HER2-positive PMGC mice compared with control treatments. Our results provide a proof-of-concept demonstration that locoregional therapy with 211At-trastuzumab may offer a new treatment option for HER2-positive PMGC.
<https://www.ncbi.nlm.nih.gov/pubmed/28514062#> 2017 Jun 27. doi: 10.1111/cas.13282
(82) Development and performance evaluation of a three-dimensional clinostat synchronized heavy-ion irradiation system
Oct 5, 2017
Hiroko IKEDA
(現所属：Gunma University Initiative for Advanced Research,
発表時の所属：Gunma University Graduate School of Medicine/Gunma University Heavy-ion Medical Center)
Outer space is an environment characterized by microgravity and space radiation, including high-energy charged particles. Astronauts are constantly exposed to both microgravity and radiation during long-term stays in space. However, many aspects of the biological effects of combined microgravity and space radiation remain unclear. We developed a new three-dimensional (3D) clinostat synchronized heavy-ion irradiation system for use in ground-based studies of the combined exposures. Our new system uses a particle accelerator and a respiratory gating system from heavy-ion radiotherapy to irradiate samples being rotated in the 3D clinostat with carbon-ion beams only when the samples are in the horizontal position. A Peltier module and special sample holder were loaded on a static stage (standing condition) and the 3D clinostat (rotation condition) to maintain a suitable temperature under atmospheric conditions. The performance of the new device was investigated with normal human fibroblasts 1BR-hTERT in a disposable closed cell culture chamber. Live imaging revealed that cellular adhesion and growth were almost the same for the standing control sample and rotation sample over 48 h. Dose flatness and symmetry were judged according to the relative density of Gafchromic films along the X-axis and Y-axis of the positions of the irradiated sample to confirm irradiation accuracy. Doses calculated using the carbon-ion calibration curve were almost the same for standing and rotation conditions, with the difference being less than 5% at 1 Gy carbon-ion irradiation. Our new device can accurately synchronize carbon-ion irradiation and simulated microgravity while maintaining the temperature under atmospheric conditions at ground level.
(83) A new system for three-dimensional clinostat synchronized X-irradiation with a high-speed shutter for space radiation research
Oct 5, 2017
Hiroko IKEDA
(現所属：Gunma University Initiative for Advanced Research,
発表時の所属：Gunma University Graduate School of Medicine/Gunma University Heavy-ion Medical Center)
Considering further human activity in space, it is necessary to study the biological effects of combined microgravity and space radiation; however, many aspects of these combined effects remain unclear. In the field of space biology, it is difficult to investigate relative biological effectiveness (RBE) and combined effects because the ability to conduct and replicate space experiments is restricted. Therefore, a new three-dimensional (3D) clinostat synchronized X-irradiation system with a high-speed shutter was fabricated following the development of a heavy-ion irradiation system. This study showed that the system could simultaneously irradiate rotating samples using the 3D clinostat, with the samples in a horizontal position. The samples were completely irradiated because of the flatness and symmetry of the irradiation fields. Doses were virtually identical under both standing and rotation conditions, with the difference being <1% under the assumption of X-irradiation at a dose of 1 Gy. Our new device could accurately synchronize X-irradiation and simulated microgravity at the ground level. The device is expected to greatly contribute to space radiation research as a valuable platform for studies concerning RBE and the combined effects of radiation under microgravity.
(84) Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets
Oct 6, 2017
Lue Sun
(Department of Radiological Health Science, Institute of Industrial Ecological Sciences,
University of Occupational and Environmental Health)
Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs) by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS) production, mitochondria function, oxygen consumption rate (OCR), energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA) resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability) in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma.