UM E-Theses Collection (澳門大學電子學位論文庫)
Title
補骨脂定通過誘導 NADPH 氧化酶 4 依賴的活性氧誘導人乳腺癌 MCF-7 細胞 DNA 損傷和自噬
English Abstract
Psoralidin (PSO) is a natural phenolic coumarin isolated from Psoralea corylifolia L. which has been proved to possess anti-cancer activities, antibacterial properties, antidepressant-like properties, anti-inflammatory activities, antifungal activities and immunomodulatory activities. It has been reported that PSO could induce cell cycle arrest, apopotosis, autophagy and inhibit metastasis in tumor. However, the mechanisms were still unclear. Our previous study indicated that PSO induced human lung cancer A549 cells autophagy in a ROS dependent manner. Here, our study focuses on the anticancer ability of PSO on MCF-7 cancer cells and the underlying mechanisms including inducing DNA damage and autophagy. MTT assay indicated that PSO exerted obviously cytotoxicity on MCF-7 cells in time- and dose-dependent manners. Meanwhile, comet assay and increased expression of DNA damage marker protein γ-H2AX expression indicated DNA damage occurred in human lung cancer A549 cells, human breast cancer MDA-MB-231 cells and MCF-7 cells. DNA damage was mediated by activation of ATM/Chk2 and ATR/Chk1 pathway in MCF-7 cells. Besides, PSO induced autophagy in MCF-7 cells evidenced by MDC fluorescence intensity increasing and the autophagic protein expressions which evidenced by PSO dose-dependent up-regulation of p62, p-ULK1 (Ser317), Beclin1, LC3 II and down-regulation of p-AKT (Ser473) and p-mTOR (Ser2448). In addition, the fluorescence probe DCFH2-DA was used to detected reactive oxygen species (ROS) levels which were enhanced to response to PSO treatment. Interestingly, both NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) could abrogate PSO-induced ROS generation, cytotoxic effects, DNA damage and autophagic pathway activation. Immunofluorescence assay and Western blot data indicated that NOX4 increased after PSO treatment. After NOX4 gene was blocked, ROS generation was reduced while PSO-induced DNA damage and autophagy were also reversed. Taken together, our study demonstrated that PSO induced DNA damage and autophagy via ROS generation in a NOX4-dependent manner.
Chinese Abstract
補骨脂定(Psoralidin,PSO)是來源于豆科植物補骨脂 Psoralea corylifolia Linn.種子的酚類天然香豆素類化合物。目前研究表明,PSO 有抗腫瘤、抗炎、 抗菌、抗抑鬱以及免疫調節等作用。PSO 能夠抑制腫瘤細胞增殖、誘導腫瘤細 胞週期阻滯、誘導腫瘤細胞凋亡以及抑制腫瘤細胞侵襲和轉移等。然而其具體 作用機制尚未闡明。我們發現 PSO 可誘導人肺癌 A549 細胞產生 ROS 依賴性自 噬。本研究主要以人乳腺癌 MCF-7 細胞為研究對象,從誘導 DNA 損傷和自噬 兩方面探討了 PSO 抗腫瘤作用的潛在機制。 MTT 實驗結果顯示 PSO 對 MCF-7 細胞存在顯著的細胞毒性,并呈時間依 賴性和濃度依賴性。彗星電泳結果顯示 PSO 處理的人肺癌 A549 細胞、乳腺癌 MCF-7 細胞和乳腺癌 MDA-MB-231 細胞出現明顯的細胞核拖尾現象。DNA 損 傷的標誌蛋白 γ-H2AX 顯著上調,提示 PSO 誘導 DNA 損傷。PSO 激活 MCF-7 細胞 DNA 損傷通路, ATM、ATR、Chk1 和 Chk2 等蛋白的磷酸化顯著上調。 MDC 染色顯示 PSO 處理的 MCF-7 細胞中的綠色熒光自噬泡明顯增多。自噬信 號通路蛋白 p62、磷酸化 ULK1(Ser317 位點)、Beclin1 和自噬標誌蛋白 LC3 II 顯著上調;AKT(Ser473 位點)和 mTOR(Ser2448 位點)蛋白表達下降,且 呈劑量依賴性。熒光探針 DCFH2-DA 檢測 ROS 發現 PSO 可誘導細胞內 ROS 的 產生。ROS 清除劑 N-乙醯基-L-半胱氨酸(N-Acetyl-L-Cysteine,NAC)和 NADPH 氧化酶抑制劑 DPI(Diphenyleneiodonium chloride)可顯著抑制 PSO 誘 導的 ROS 生成、細胞毒性作用、DNA 損傷應答和自噬通路的激活。WB 和免疫 熒光結果顯示 PSO 顯著上調 NOX4 蛋白表達。NOX4 siRNA 沉默 NOX4 后, PSO 誘導的 ROS 生成、DNA 損傷和自噬被部分逆轉。 綜上所述,本研究顯示補骨脂定誘導腫瘤細胞產生 DNA 損傷和自噬,其 機制可能是通過誘導 NOX4 依賴的活性氧生成。 關鍵字:補骨脂定,DNA 損傷,自噬,NOX4,ROS,MCF-7
Issue Date
2015
Author
任國文
Faculty
Institute of Chinese Medical Sciences
Degree
M.Sc.
Subject
Cancer -- Treatment
癌症 -- 治療
Antineoplastic agents -- China
抗惡性增生腫瘤藥物 -- 中國
Medicinal plants -- China -- Analysis
藥用植物 -- 中國 -- 化學分析
Supervisor
陳修平
Files In This Item:
Full-text (Intranet only)
Location
1/F Zone C
Supervisor
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