| Project/Area Number |
12671372
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cerebral neurosurgery
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| Research Institution | University of the Ryukyus |
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Principal Investigator |
YOSHII Yoshihiko University of the Ryukyus, Neurosurgery, Professor, 医学部, 教授 (50110507)
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| Co-Investigator(Kenkyū-buntansha) |
NOSAKI Masatosi Okinawa Prefectural Institute of Health and Environment, Health Science, Chairman Instructor, ハブ研究室, 室長
SUN L.K. University of the Ryukyus, Neurosurgery, Assistant Professor, 医学部, 助手 (40325840)
SAITO Atsushi University of the Ryukyus, Neurosurgery, Associate Professor, 医学部附属病院, 講師 (40305199)
鶴嶋 英夫 琉球大学, 医学部, 助手 (50315470)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Okinawa Habu venom / L-aminoacid oxidase / metalloprotease / vascular endothel / glioma cell / apoptosis / oxidative stress / OHAP-1 / L-aminoacid oxidase / 移植腫瘍 / グリオーマ / 抗腫瘍効果 / ハブ毒 / アミノ酸構造 / OHAP-I / OHAP-II / クリオーマ / 皮下移植腫瘍 / プロティン / 殺細胞効果 |
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| Research Abstract |
Okinawa Habu (Trimeresurus Flavoviridis) venom is well known for its toxic efficacy, from which one kind of specific protein. Okinawa Habu apoxin protein-1 (OHAP-1) has been extracted. The propose of this study was to investigate whether OHAP-1 could induce apoptosis in some glioma and endothelial cells, and if so elucidate the possible mechanism involved. Two normal and three malignant glioma cell lines were tested. The malignant glioma cell lines were rat C6 and human RBR 17T, U251. OHAP-1 inhibited growth of all cell lines. Whether or not the apoptosis had been induced was determined by using DNA gel electrophoresis, DNA flow cytometry and TUNEL assay. After OHAP-1 treatment, DNA fragmentation, an increase in the percentage of subdiploid DNA content, and TUNEL positive cells were found in the C6, RBR17T, and U251 cells. Furthermore, OHAP-1 showed L-amino acid oxidase (LAAO) activity. In order to study the mechanism of apoptosis induced by OHAP-1, the changes of intracellular reactiv
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e oxygen species (ROS) were measured using flow cytometry, and the expression of p53 protein was examined using immunohistochemistry. OHAP-1 was found to generate ROS and increase the expression of p53 protein in glioma cells. The inhibiting effect of OHAP-1 on three tested cells was reversed when an antioxidant of either catalase or reduced glutathione (GSH) was added; its apoptotic effect correspondingly became weaker. In this study, the apoptotic effect of OHAP-1 on some malignant glioma and transformed vascular endothelial cells was confirmed, and it could be that this effect might be mediated through promoting the generation of intracellular ROS and p53 protein expression in glioma cells. It was suggested that OHAP-1 is promising as a potential candidate for clinical tumor therapy.
In order to study the therapeutic efficacy of OHAP-1 for treatment of malignant brain tumors, an animal tumor model was used in this examination. In the tumor transplanted subcutaneously the C6 glioma cells to Wistar rats, tumor growth inhibited after 5 days treatment of OHAP-1. In addition, the apoptotic index of tumor tissues assessed by the TUNEL method was significantly increased, and angiogenesis decreased in rat after 7 days treatment with OHAP-1. These results suggest that the OHAP-1 may have a protective effect of tumor-growth inhibition in rats through the induction of apoptosis. Because we extracted a specific protein from Okinawa Habu snake venom which we named "Okinawa Habu apoxin protein-2 (OHAP-2)" and its cytotoxic activity was strongest estimated by the cell viability assay by MTT method, we reported the biological characteristics of OHAP-2. Human and rat malignant glioma cell lines (RBR17T and C6) and ECV 304 cell lines were used in this study. The ladder formation of the fragmented DNA and TUNEL positive cells in the cells treated with OHAP-2. OHAP-2 showed a striking homology to HR2a (Amami Habu venom) and also has a biologically metalloprotease activity as well as high protease activity and snake metalloprotease cuts off the anchor of the pro-inflammatory cytokine TNF-α. Therefore, in glioma therapy OHAP-2 is feasible to suggest that released soluble Fas L and/or TNF-a induces the Fas-mediated (Fas/APO-1L) killing and/or TNF related apoptosis by TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in paracrine fashion; but this remains a matter for further study. Less
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