The effects of peanut stilbenoids on HL-60 cell line

二苯乙烯類 (stilbenoids)為植物抵抗黴菌或病蟲害的天然抗生素，許多研究報告指出，二苯乙烯類具有抗氧化、抗發炎、防癌及抗癌等生理活性。本論文研究純化自花生仁的二苯乙烯類，包括resveratrol (RSV)、arachindin-1 (Ara-1)、arachindin-3 (Ara-3)及isopentadienylresveratrol (IPD)的抗癌活性，以HL-60血癌細胞株當作實驗模型，研究中發現，RSV、Ara-1及Ara-3可抑制HL-60細胞株的生長，其中，Ara-1在較低的濃度就可以抑制細胞生長 (EC50為4.2 μM)。以Ara-1處理HL-60細胞，在短時間內可造成細胞粒線體膜電位的下降、caspases的活化、DNA斷裂以及細胞週期改變。Ara-3處理HL-60細胞則是觀察到其細胞膜完整性的改變、細胞粒線體膜電位的下降，以及caspases的活化。綜合以上結果，Ara-1及Ara-3處理HL-60細胞皆可以引發細胞走向凋亡，而對正常不增生的周邊血單核球細胞則無影響，因此具有成為抗癌藥物的潛力。由於體外試驗Ara-1可抑制Topoisomerase II的活性，但對HL-60與HL-60/MX2 (Topoisomerase II缺乏的HL-60突變株)的毒性沒有差異，推測Ara-1造成細胞走向凋亡並非透過抑制Topoisomerase II。但仍可由以上結果顯示，Ara-1及Ara-3可能具有抗癌的潛力。Stilbenoids are phytoalexins produced by plants under attack by pathogens such as fungi. Previous sutdies have demonstrated that stilbenoids have bioactivities such as anti-oxidation, anti-inflammation, cancer chemoprevention and anti-carcinogenic effect. In this study, resveratrol (RSV), arachidin-1 (Ara-1), arachidin-3 (Ara-3), and isopentadienylresveratrol (IPD) were isolated from peanuts. RSV, Ara-1 and Ara-3 display cytotoxic effect on HL-60, and Ara-1 is the most toxic. Further study showed that Ara-1 treatment on HL-60 cells caused loss of mitochondrial membrane potential (MMP), caspases activation, and changes in cell cycle distribution. Ara-3 treatment on HL-60 cells caused loss of MMP and cell membrane integrity as well as caspases activation. These results suggested that Ara-1 and Ara-3 are potential anticancer agents that induced cancer cell apoptosis. Ara-1 and RSV are cytotoxic to proliferating cells, HL-60, but did not affect on nonproliferating human peripheral blood mononuclear cells. Furthermore, Ara-1 was found to inhibit the activity of topoisomerase II in vitro, but showed no significant difference in cytotoxicity between HL-60 and Topo II defective HL-60/MX2 cells. Topoisomerase II inhibition may not be involved in the mechanisms of apoptosis induction by Ara-1 in HL-60 cells. These results indicate that Ara-1 and Ara-3 are potential anti-cancer agents.中文摘要………………………………………………………………………………4文摘要………………………………………………………………………….......5寫表…………………………………………………………………………………6、序論……………………………………………………………………………….7.1花生中的二苯乙烯類……………………………………………………….........7.2二苯乙烯類相關研究…………………………………………………………….7.3 癌症的治療…………………………………………………….........................8.4 細胞凋亡 (Apoptosis) ………………………................................................9.5 細胞週期 (Cell cycle) …………………………………………………..........11.6 拓樸異構酶II (Topoisomerase II) …………………………………………....12.7 研究目的……………………………………………......................................13、材料與方法………………………………………………………………..........14.1花生中二苯乙烯類的純化及穩定性探討……………………………………...14.2細胞培養………………………………………………………………………...14.3人類周邊血單核球細胞 (PBMC)的分離…………………………………......15.4細胞活性測定…………………………………………………………………...15.5細胞粒線體膜電位的測定……………………………………………………...16.6細胞內活性氧自由基 (reactive oxygen species; ROS)測定……………....16.7細胞DNA含量分析………………………………………………………….....16.8細胞膜完整性分析……………………………………………………………...16.9細胞內Caspases活性的測定………………………………………………....17.10 Topo II活性的測定…………………………………………………………....17、結果………………………………………………………………………….......18.1 花生中二苯乙烯類的純化……………………………………………………..18.2 Resveratrol、arachidin-1及arachidin-3能夠抑制HL-60細胞生長…….......18.3 Arachidin-1及resveratrol抑制人類血癌細胞株HL-60的生長而對不增生的PBMC則無影響……………………………………………..................................18.4 Arachidin-1處理引發HL-60細胞凋亡………………………………………..19.5 Arachidin-3處理引發HL-60細胞凋亡………………………………………..21.6 Arachidin-1及resveratrol抑制Topo II 活性………………………………...22.7 Arachidin-1不會與Topo II直接結合而抑制其活性………………………….22.8 Arachidin-1及arachidin-3對人類血癌細胞株HL-60與HL-60/MX2的毒性沒有差異……………………………………………………………………............23.9 Arachidin-1、arachidin-3及Isopentadienylresveratrol穩定性探討……...23、討論……………………………………………………………………….........24、參考文獻………………………………………………………………………..52品清單………………………………………………………………………........5