Apoptotic effect in the glioma cells induced by specific protein extracted from Okinawa Habu ( Trimeresurus flavoviridis) venom in relation to oxidative stress

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 purpose of this study was to investigate whether OHAP-1 could induce apoptosis in some glioma cells, and if so, to elucidate the possible mechanism involved. 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 reactive 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 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.