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

冲绳蛇毒(Trimeresurus Flavoviridis)以其毒力著称，其中有一种特殊的蛋白质。冲绳哈布Apooxin蛋白-1(OHAP-1)已被提取。本研究旨在探讨OHAP-1是否能诱导某些胶质瘤和血管内皮细胞的凋亡，并阐明其可能的作用机制。检测了2个正常胶质瘤细胞系和3个恶性胶质瘤细胞系。恶性胶质瘤细胞系为大鼠C6和人RBR 17T、U251。OHAP-1对所有细胞株的生长均有抑制作用。用DNA凝胶电泳法、DNA流式细胞术和TUNEL法检测细胞是否诱导了细胞凋亡。经OHAP-1处理后，C6、RBR17T和U251细胞DNA片段化，亚二倍体DNA含量增加，TUNEL阳性细胞数增加。此外，OHAP-1还具有L氨基酸氧化酶(LAAO)活性。为探讨OHAP-1诱导细胞凋亡的机制，细胞内反应性…的变化用流式细胞仪检测细胞内氧自由基(ROS)，用免疫组织化学方法检测P53蛋白的表达。研究发现，OHAP-1可在胶质瘤细胞中产生ROS并增加P53蛋白的表达。当加入抗氧化剂过氧化氢酶或还原型谷胱甘肽(GSH)时，OHAP-1对三个受试细胞的抑制作用被逆转；其诱导凋亡的作用相应减弱。本研究证实了OHAP-1对某些恶性胶质瘤和转化的血管内皮细胞的促凋亡作用，其机制可能是通过促进细胞内ROS和P53蛋白表达来实现的。为了研究OHAP-1对恶性脑肿瘤的治疗效果，本实验采用动物肿瘤模型。在Wistar大鼠皮下移植C6胶质瘤细胞的肿瘤中，OHAP-1治疗5天后肿瘤生长受到抑制。此外，OHAP-1治疗7d后，TUNEL法检测肿瘤组织的凋亡指数明显增加，血管生成减少。这些结果提示，OHAP-1可能通过诱导细胞凋亡而对大鼠肿瘤生长具有保护作用。由于我们从冲绳哈布蛇毒中提取了一种特异性蛋白，命名为OHAP-2(OHAP-2)，并通过四甲基偶氮唑盐比色法测定其细胞活性，因此我们报道了OHAP-2的生物学特性。本研究以人和大鼠恶性胶质瘤细胞系(RBR17T和C6)和ECV 304细胞系为研究对象。经OHAP-2处理后，细胞DNA片段化和TUNEL阳性细胞呈梯状分布。OHAP-2与Amami Habu蛇毒HR2a具有显著的同源性，并且具有较高的生物学活性和金属蛋白酶活性，蛇金属蛋白酶切断了促炎细胞因子α的锚定。因此，在脑胶质瘤治疗中，OHAP-2提示释放可溶性Fas、L和/或肿瘤坏死因子-α通过旁分泌方式诱导肿瘤坏死因子相关的凋亡诱导配体(TRAIL)介导的Fas/APO-1L杀伤和/或肿瘤坏死因子相关的细胞凋亡是可行的，但这还有待进一步研究。较少

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