| Summary: | 碩士 === 嘉南藥理科技大學 === 生物科技系暨研究所 === 99 === Epstein-Barr virus (EBV) is a gammaherpesvirus and is associated with pathogenesis of lymphoid and some epithelial tumors. The root of Polygonum cuspidatum is widely used as a medicinal herb in Asia. Previous studies showed the ethanolic extract and ethyl acetate subfraction F3 from Polygonum cuspidatum root inhibit the functions of EBV latent membrane protein 1 and promote apoptosis of EBV-positive tumor cells. The purpose of this study is to investigate the subfraction F2a of ethyl acetate from Polygonum cuspidatum root induces apoptosis of EBV-positive tumor cells via a reactive oxygen species (ROS) mechanism. The viability of EBV-positive tumor cells line, B95-8 was determined by MTT. The expression of LMP1, EBNA1, EBV lytic proteins and apoptotic-related proteins were determined by western blotting. Real-time PCR was used to measure the EBV DNA copy number. Fluorescence DNA-binding dye, DAPI, was used to detect the nuclear chromatin morphology. Comet assay was used to assess the extent of DNA damage. Flow cytometry analysis was used for the detection of reactive oxygen species and percentage of apoptotic cells. Results showed that F2a promotes the expression of EBV LMP1, EBNA1, and lytic proteins, and EBV DNA copy number increasing which induced high level of reactive oxygen species (ROS) in EBV-positive cells. Furthermore, it also induces high level of ROS in EBV-positive cells. The high level of reactive oxygen species leads to the damage of DNA, activation of the apoptotic protein caspase 3 and the cleavage of PARP, which promoted EBV-positive tumor cells exhibiting typical features of apoptosis-chromatin condensation, nuclear fragmentation and the percentage of apoptotic cells increasing. These findings suggest that the F2a induced apoptosis of EBV-positive tumor cells may mediated by the high level of ROS production, which maybe used as a useful therapeutic drug in the treatment of EBV-positive tumor cells.
|
|---|
