EB 病毒為人類皰疹病毒，與導致淋巴細胞和上皮細胞腫瘤有相關性。虎杖是一種在亞洲被廣泛應用的中草藥，以前研究發現虎杖乙醇萃取物及虎杖根部乙酸乙酯分離物 F3 能夠干擾 EB 病毒潛伏期膜蛋白質 1 (Latent membrane protein，LMP1) 功能，使含有 EB 病毒的腫瘤細胞走向凋亡，所以本研究進一步探討虎杖根部乙酸乙酯分離物 F2a 是否可藉由誘發活性氧 (Reactive oxygen species，ROS) 訊息傳遞使 EB 病毒腫瘤細胞走上凋亡。實驗以 MTT 測定 EB 病毒腫瘤細胞株 B95-8 細胞的存活率；西方墨點法測定 EB 病毒潛伏期膜蛋白質 1、EB 病毒核抗原 1 、EB 病毒溶裂期蛋白質及凋亡相關蛋白質的表現；利用即時定量聚合酶連鎖反應測定 EB 病毒 DNA 的複製量；利用彗星試驗觀察 DNA 損傷程度；使用螢光 DNA 結合染劑-DAPI 觀察細胞核型態；使用流式細胞儀測定細胞內 ROS 含量的變化及早期和晚期凋亡的百分比。結果顯示虎杖根部乙酸乙酯分離物 F2a 會增加 EB 病毒潛伏期膜蛋白質 1、EB 病毒核抗原 1、EB 病毒溶裂期蛋白質的表現及增加 EB 病毒 DNA 複製，使得細胞內 ROS大量增加。高量 ROS 會增加造成細胞 DNA 損傷，活化凋亡蛋白質 caspase-3及並導致 PARP 的裂解，使早期及晚期凋亡百分比有增加。這些結果證明 F2a 可以誘發 EB 病毒腫瘤細胞內 ROS 產生，進而誘發細胞的凋亡，可以 F2a 發展成為治療 EB 病毒相關腫瘤的藥劑。 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.
