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Title: 固形腫瘍の再酸素化における一酸化窒素の役割とその生成機構
Other Titles: The mechanism of radiation-induced endothelial nitric oxide synthase activation and its role in tumor reoxygenation after X-irradiation
Authors: 永根, 大幹1 Browse this author
Authors(alt): Nagane, Masaki1
Issue Date: 25-Mar-2014
Abstract: Low oxygen conditions drastically decreases cellular radiosensitivity and facilitate the adaptive responses of tumor. Hence, hypoxic regions in solid tumor are well-known obstacle for successful radiotherapy. It occurs mainly as a result of the excess proliferation of tumor cells and accompanying deficiency in blood and nutrient supplies. It has been reported that ionizing radiation (IR) decreases hypoxic regions in tumor, leading to the improvement of radiosensitivity of solid tumor. However, the mechanism of IR-induced tumor reoxygenation has not been elucidated. In the first study, to clarify the effect of radiation-induced nitric oxide (NO) on tumor oxygenation status and the NOS isoforms responsible for the NO production after IR, transplanted SCCVII tumor was employed. Tumor oxygenation status after IR was evaluated by in vivo electron spin resonance method with the oxygen sensitive probe, immunohistochemical technique with two different hypoxia probes, and Hoechst33342 perfusion assay. It was shown that IR increased tissue pO2 and tissue perfusion after IR. In addition, decrease of the hypoxic regions in tumor was observed after IR. To clarify the involvement of NO in this reoxygenation, a wide-range NOS inhibitor L-NAME was administered to tumor-bearing mice. L-NAME treatment significantly attenuated reoxygenation in hypoxic regions after IR. These results suggested that IR increased NO production that increased tissue perfusion in tumor, leading to the tumor reoxygenation. In mammalian cells, there are three NOS isoforms, endothelial NOS (eNOS), inducible NOS (iNOS), and neural NOS (nNOS). To determine the NOS isoforms responsoble for NO-induced reoxygenation, RT-qPCR analysis, Western blot analysis, and NOS activity assay were used. RT-qPCR analysis showed that nNOS hardly expressed in SCCVII tumor, and that IR did not increase mRNA of NOS isoforms. Western blot analysis showed IR did not increase protein expression of iNOS and eNOS. NOS activity assay revealed that IR increased NOS activity, and iNOS was not involved in this activity. In addition, immunohistochemistry showed that eNOS was expressed in vascular endothelial cells of tumor. These results suggested that IR increased eNOS activity in vascular endothelial cells, leading to the tumor reoxygenation. However, the mechanism of eNOS activation after IR remains unclear. In the second study, bovine aortic endothelial cells (BAEC) were employed to investigate the mechanisms eNOS activation in vitro after IR. As a mechanism of eNOS activation, phosphorylation of eNOS-Ser1179 by Ser/Thr kinases is a critical step. IR is known to induce DNA-double strand breaks (DSBs) and elicit subsequent various adaptive responses toward IR. Ataxia telangiectasia mutated (ATM) plays an important role in coordinating the cellular response to DSBs. In addition to kinases, HSP90 has also been identified as an important regulator modifying ATM function after IR. In this study, I aimed to clarify the involvement of DNA-damage response in IR-induced eNOS activation. NOS activity assay showed that IR increased NOS activity at 12 and 24 h after IR. In these time points, eNOS-Ser1179 phosphorylation was increased. ATM phosphorylation was increased at 0.5 h after IR, and lasted to 24 h. An ATM inhibitor Ku-60019 inhibited eNOS and ATM phosphorylation after IR. NOS activity was attenuated by Ku-60019. Similarly, a HSP90 inhibitor geldanamycin inhibited ATM and eNOS phosphorylation, and eNOS activation. These results suggested that IR increased eNOS activity through HSP90/ATM pathway. In conclusion, this study provides new insight for mechanisms in radiation-induced tumor reoxygenation. IR increases eNOS activity via DNA damage response. Activation of eNOS increases tissue perfusion through the production of NO and reoxygenates the hypoxic regions in solid tumor. This radiation-induced tumor reoxygenation increases tumor radiosensitivity toward subsequent irradiation, providing the new insight for the improvement of radiotherapy by modulating eNOS activity.
Conffering University: 北海道大学
Degree Report Number: 甲第11282号
Degree Level: 博士
Degree Discipline: 獣医学
Examination Committee Members: (主査) 教授 稲波 修, 教授 木村 和弘, 准教授 高木 哲, 准教授 山盛 徹
Degree Affiliation: 獣医学研究科(獣医学専攻)
Type: theses (doctoral)
Appears in Collections:学位論文 (Theses) > 博士 (獣医学)
課程博士 (Doctorate by way of Advanced Course) > 獣医学研究科(Graduate School of Veterinary Medicine)

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