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Title

葫蘆素 E 抑制肺癌 95D 細胞增殖的作用與機制研究

English Abstract

Cucurbitacins is a kind of highly oxidized tetracyclic triterpenoids. Modern researches have shown that cucurbitacins (such as cucurbitacin B) have potent antitumor acitvities both in vitro and in vivo, they could also induces cell cycle arrest and apoptosis on cancer cells. Cucurbitacin E (CuE) is one of tetracyclic triterpenoid compounds which is one of the most abundant members of the cucurbitacins’ family. It has extensive biological activities, such as liver protection, anti-inflammatory, immunoregulation effects, anti-tumor and so on. But nowadays the anti-tumor effect and the detail mechanisms of CuE remain to be clear. This study mainly focused on the antiproliferation effect induced by CuE on 95D lung cancer cells and also its possible mechanisms. The cell viability and proliferation was measured by MTT assay and colony formation assay. Intracellular ROS formation was determined by a fluorescent probe DCFH2-DA with flow cytometry. JC-1 staining was used to analyze the change of mitochondrial membrane potential induced by CuE. The apoptosis induced by CuE was detected by TUNEL. Flow cytometry analyzed the effect on cell cycle induced by CuE. AND MDC staining was used for detecting formation of autophagic vacuoles. Western blotting was used to determine the expression of some apoptosis, autophagy, endoplasmic reticulum stress and cell cycle related proteins. The results indicated that CuE significantly inhibited cell viability and proliferation of lung cancer 95D cells in a dose- and time- dependent manner. CuE induced the generation of ROS on 95D cells remarkably, and it was reversed by NAC. CuE decreased the mitochondrial membrane potential. NAC, Z-VAD-FMK and AcDEVD-CHO partial reversed the results. CuE induced apoptosis on 95D cells, and decrease BCl-2. CuE influenced autophagy Marker protein LC-3B on a time- and dose-dependent manner and also down regulated SQSTM1/p62, induced the formation of autophagic vacuoles. In addition, CuE significantly induced G2 / M phase arrest and downregulate Cyclin B1 and cdc25C on 95D cells. In conclusion, the present study indicates that CuE could inhibit viability and proliferation of 95D cells by inducing apoptosis, autophagy and G2/M phase arrest, and its mechanisms may be related to the generation of ROS and regulation of the proteins.

Chinese Abstract

葫蘆素類(Cucurbitacins)是一類四環三萜類化合物,廣泛分佈於包括葫蘆科 在內的多種植物,也是傳統中藥瓜蒂的主要藥效成分。現代研究表明,葫蘆素 類化合物(如葫蘆素 B)在體內外都具有較好的抗腫瘤活性,可誘導腫瘤細胞發生 週期阻滯和凋亡。 葫蘆素 E (Cucurbitacin E, CuE)是葫蘆素家族中含量最豐富的成員之一,具 有保肝、抗炎、抗菌、抗腫瘤等活性。但目前對其抗腫瘤的作用與機制研究尚 不深入。本研究以肺癌 95D 細胞株為研究對象,探討 CuE 抑制 95D 細胞的增殖 作用及其可能的機制。採用 MTT 法和克隆形成實驗分別測定了 CuE 處理後對 細胞活力和增殖的影響;以螢光探針 DCFH2-DA、流式細胞儀測定了 CuE 對細 胞內活性氧(Reactive Oxygen Species, ROS)的作用;螢光探針 JC-1 染色分析 CuE 對線粒體膜電位的影響;TUNEL 法檢測了 CuE 對細胞凋亡的影響;流式 細胞儀測定了 CuE 對細胞週期的影響;以 MDC 染色檢測了 CuE 誘導細胞內自 噬泡的形成;Western Blot 測定了細胞週期、凋亡、自噬和內質網應激相關蛋白 質的表達。結果顯示,CuE 顯著抑制肺癌細胞 95D 的活力和增殖,且呈濃度和 時間依賴性;CuE 顯著誘導 95D 細胞內 ROS 生成,並可被 ROS 清除劑 N-乙醯 基-L-半胱氨酸(N-Acetyl-L-Cysteine, NAC)所抑制;CuE 降低細胞線粒體膜電位; NAC、Caspase 抑制劑 Z-VAD-FMK 以及 Caspase3 抑制劑 Ac-DEVD-CHO 可部 分逆轉 CuE 誘導的死亡;CuE 可以誘導 95D 細胞凋亡,下調 Bcl-2 的蛋白表達; CuE 還可以時間劑量依賴的誘導細胞自噬 Marker 蛋白 LC-3B 的表達、下調 SQSTM1/p62 的表達,誘導細胞內自噬泡形成。此外,CuE 顯著誘導 95D 細胞 G2/M 期阻滯並下調 Cyclin B1、cdc25C。綜上所述,本實驗表明,CuE 可通過 誘導 95D 細胞 G2/M 期阻滯、凋亡以及自噬抑制 95D 細胞增殖,其機制可能與 誘導 ROS 生成以及調節相關蛋白表達有關。 關鍵字:葫蘆素 E 活性氧 凋亡 自噬 內質網應激 細胞週期

Issue date

2015

Author

馬桂鑫

Faculty

Institute of Chinese Medical Sciences

Degree

M.Sc.

Subject

Lungs -- Cancer -- Treatment

肺臟 -- 癌症 -- 治療

Medicinal plants -- China

藥用植物 -- 中國

Chinese Medicinal Science -- Institute of Chinese Medical Sciences

中藥學 -- 中華醫藥研究院

Supervisor

陳修平

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