Apoptotic effect by specific proteins extracted from Okinawa Habu (Trimeresurus Flavoviridis) snake venom into glioma and vascular endothelial cells

从冲绳竹叶青蛇毒中提取的特定蛋白质对神经胶质瘤和血管内皮细胞的细胞凋亡作用

• 批准号: 12671372
• 负责人: YOSHII Yoshihiko
• 金额: $ 1.98万
• 依托单位: University of the Ryukyus
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671372/
• 关键词: 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

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 … More 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

冲绳哈布（Trimeresurus Flavoviridis）毒是一种毒性很强的毒，其中含有一种特异性蛋白质。提取了冲绳哈布脱辅基蛋白-1（OHAP-1）。本研究旨在探讨OHAP-1是否能诱导胶质瘤细胞和血管内皮细胞凋亡，并探讨其可能的作用机制。两个正常和三个恶性胶质瘤细胞系进行了测试。恶性胶质瘤细胞系为大鼠C6和人RBR 17 T、U251。OHAP-1抑制所有细胞系的生长。采用DNA凝胶电泳、DNA流式细胞术和TUNEL法检测细胞凋亡情况。OHAP-1处理后，C6、RBR 17 T和U251细胞中DNA片段化，亚二倍体DNA含量百分比增加，TUNEL阳性细胞。此外，OHAP-1显示L-氨基酸氧化酶（LAAO）活性。为探讨OHAP-1诱导细胞凋亡的机制，采用流式细胞术检测OHAP-1诱导细胞凋亡的细胞内反应性变化， ...更多信息 流式细胞术检测细胞内氧自由基（ROS）水平，免疫组化法检测p53蛋白表达。OHAP-1可诱导神经胶质瘤细胞产生活性氧，并增加p53蛋白的表达。当加入过氧化氢酶或还原型谷胱甘肽（GSH）抗氧化剂时，OHAP-1对三种受试细胞的抑制作用被逆转，其凋亡作用相应地减弱。本研究证实了OHAP-1对某些恶性胶质瘤和转化血管内皮细胞的凋亡作用，这种作用可能是通过促进胶质瘤细胞内ROS的产生和p53蛋白的表达而介导的。为了研究OHAP-1对脑恶性肿瘤的治疗作用，本研究采用动物肿瘤模型，观察OHAP-1对脑恶性肿瘤的治疗作用。在皮下移植C6神经胶质瘤细胞至Wistar大鼠的肿瘤中，OHAP-1处理5天后肿瘤生长受到抑制。此外，用OHAP-1处理7天后，通过TUNEL方法评估的肿瘤组织的凋亡指数显著增加，并且大鼠中的血管生成减少。这些结果表明，OHAP-1可能通过诱导细胞凋亡对大鼠肿瘤生长抑制具有保护作用。由于我们从冲绳哈布蛇毒中提取了一种特异性蛋白质，命名为“冲绳哈布脱辅基蛋白-2（OHAP-2）”，MTT法测定其细胞毒活性最强，因此我们报道了OHAP-2的生物学特性。本研究使用人和大鼠恶性胶质瘤细胞系（RBR 17 T和C6）和ECV 304细胞系。OHAP-2处理细胞后DNA片段和TUNEL阳性细胞的梯状条带形成。OHAP-2显示出与HR 2a（Amami Habu venom）惊人的同源性，并且还具有生物学金属蛋白酶活性以及高蛋白酶活性，并且蛇金属蛋白酶切断促炎细胞因子TNF-α的锚。因此，在胶质瘤治疗中，OHAP-2可能提示释放的可溶性Fas L和/或TNF-α以旁分泌方式通过TRAIL（肿瘤坏死因子相关的凋亡诱导配体）诱导Fas介导的（Fas/APO-1 L）杀伤和/或TNF相关的凋亡，但这仍有待于进一步研究。少

项目成果

Yoshii Y, Sun LK, Hyodo A, Sugimoto K, Saito A, Kariya K, Nozaki M, Morine N: "Proteins containing metalloprotease activity from Okinawa Habu venom induce apoptosis in glioma cells"Toxicon. (in press). (2003)

Yoshii Y、Sun LK、Hyodo A、Sugimoto K、Saito A、Kariya K、Nozaki M、Morine N：“来自冲绳 Habu 毒液的含有金属蛋白酶活性的蛋白质可诱导神经胶质瘤细胞凋亡”Toxicon。

孫連坤: "ラットC6グリオーマモデルにおける沖縄ハブ毒素由来のOHAPIの腫瘍増殖抑制効果"日本脳腫瘍カンファランス講演集. (2002)

孙连坤：“冲绳枢纽毒素OHAPI对大鼠C6神经胶质瘤模型的肿瘤生长抑制作用”日本脑肿瘤会议论文集（2002）。

Lian-Kun Sun: "Proteins containing L-amino acid oxidase activity from Okinawa Habu venom induce apoptosis in glioma cells"Toxicology in Vitro. (in press). (2003)

孙连坤：“冲绳哈布毒液中含有 L-氨基酸氧化酶活性的蛋白质诱导神经胶质瘤细胞凋亡”体外毒理学。

Lian-kun Sun: "Apoptotic effect by specific proteins extracted from Okinawa Habu venom into the glioma cells"J Neuro-oncology. (in press). (2001)

孙连坤：“从冲绳哈布毒液中提取的特定蛋白质对神经胶质瘤细胞的凋亡作用”J Neuro-oncology。

Yoshihiko Yoshii: "Proteins containing metall oprotease activity from Okinawa Habu venoin induce apoptosis in glioma cells"J Neuro-oncol. (in press). (2002)

Yoshihiko Yoshii：“来自冲绳 Habu 静脉的含有金属蛋白酶活性的蛋白质可诱导神经胶质瘤细胞凋亡”J Neuro-oncol。