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  • 學位論文

葫蘆素E誘導人類鼻咽和咽癌細胞株細胞週期G2/M 停滯及誘發細胞自噬之探討

The study of cucurbitacin E induces G2/M arrest and autophagy in human nasopharyngeal and pharynx carcinoma cells

指導教授 : 徐怡強

摘要


惡性腫瘤是疾病十大死因的第一名,可見癌症的預防與治療重要性與日俱增。近年來許多研究發現天然植物或食療性素材中具有生物活性的成分同時也有較強的化學預防性功效(chemopreventive),並且可能具有癌症化學治療的作用。 在本論文中葫蘆素E (Cucurbitacin E; CuE)是萃取自葫蘆科一年生蔓性草本植物甜瓜(Cucumic melo L.)的瓜蒂與果柄,傳統上瓜蒂(葫蘆素E)用來治療溼熱毒所導致的慢性遷延性肝炎、慢性肝炎及原發性肝癌的輔助治療,而在本論文探討葫蘆素E對人類鼻咽和咽癌細胞的抗腫瘤生長的作用。利用體外實驗來探討葫蘆素E的抗腫瘤生長效果,分別使用人類鼻咽癌細胞(Hone-1)和人類咽癌細胞(Detroit 562)細胞株進行分析。 利用MTT assay進行細胞劑量依賴性測試,經葫蘆素E加藥處理人類鼻咽和咽癌細胞24至72小時後,觀察到明顯的劑量依賴性,並利用流式細胞儀發現配合Propidium Iodide 染色以及Annexin V 加上 Propidium Iodide 雙染色測試經葫蘆素E處理後觀察細胞週期的變化和細胞是否進行細胞凋亡,實驗結果發現人類鼻咽癌細胞經葫蘆素E處理後造成細胞週期G2/M phase停滯和葫蘆素E不會造成人類鼻咽和咽癌細胞的細胞凋亡作用。並以MPM-2-FITC偵測經葫蘆素E處理人類鼻咽和咽癌細胞的有絲分裂期的變化量,發現隨著劑量的增加有絲分裂期的變化量有增加的趨勢,有絲分裂期表現量的增加意謂著調控G2/M相關蛋白質有受到葫蘆素E的影響,利用Western blot(西方墨點法)偵測調控G2/M相關蛋白質Cyclin B1和CDC2的表現,實驗結果發現人類鼻咽癌細胞經葫蘆素E處理後Cyclin B1和CDC2的基因表現量有下降的趨勢,為了更進一步了解有關Cyclin B1和CDC2的上游調控,我們利用Microarray的分析,並從KEGG pathway 上比對發現有關調控Cyclin B1和CDC2的上遊基因GADD45 family(GADD45α、GADD45β、GADD45γ)的表現是增加的,並以RT-PCR和Q-PCR分析Cyclin B1、CDC2和GADD45 family(GADD45α、GADD45β、GADD45γ),實驗結果發現人類鼻咽癌細胞經葫蘆素E處理後Cyclin B1和CDC2的基因表現是下降的,而GADD45 family(GADD45α、GADD45β、GADD45γ)的表現有增加的趨勢。 本研究利用葫蘆素E作為有效的頭頸癌治療(或輔助化學治療)用藥物,並且同時深入探討其對於人類頭頸癌之抑癌作用機轉與細胞週期及癌化、抑癌化基因蛋白質表現之調控方式,可以提供頭頸癌治療及預防一個新的方向。

並列摘要


Cancer is the leading cause of death in the developed world, which shows the growing importance of cancer prevention and treatment. In recent years, research has shown that certain natural plants and dietotherapeutic materials have bioactive components, display stronger chemopreventive effects, and may even be used in chemotherapy. We extracted Cucurbitacin E (CuE) from the stalk and stem of Cucumic melo L., an annual tendrillar herbaceous plant belonging to the Cucurbitaceae family. Traditionally, the stem (CuE) is used in the adjuvant therapy of the chronic persistent hepatitis, chronic hepatitis, and primary liver cancer caused by damp heat. In this study, we investigated the effectiveness of CuE in preventing the growth of human nasopharynx and pharynx cancer cells. We performed in vitro experiments using the Hone-1 and Detroit 562 cell lines for pharynx cancer and nasopharynx cancer, respectively. We employed MTT assays to gauge the dose-dependence of the cells. Those treated with CuE displayed significant dose-dependence after 24 to 72 hours. We then used a flow cytometer in conjunction with propidium iodide staining and Annexin V/propidium iodide double staining to look for changes in cell cycles and apoptosis after the CuE treatment. The results revealed that the CuE treatment caused a stagnation of cells in the G2/M phase but did not lead to apoptosis. Subsequently, we used MPM-2-FITC to detect the number of nasopharynx and pharynx cancer cells treated with CuE that underwent mitosis, which was found to increase with the dosage. The increase in motisis expression means that CuE affects the protein associated with the regulation of the G2/M phase. We thus used the western blot to detect the expression of Cyclin B1 and CDC2, two proteins associated with the regulation of the G2/M phase. The results show that CuE treatment decreases the gene expression of these two proteins in human nasopharynx and pharynx cancer cells. To further understand the upstream regulatory effects of Cyclin B1 and CDC2, we used microarray analysis and compared KEGG pathways, thereby establishing the increased expression of the upstream genes, the GADD45 family (GADD45α, GADD45β, GADD45γ). Using RT-PCR and Q-PCR to analyze Cyclin B1, CDC2, and the GADD45 family (GADD45α, GADD45β, GADD45γ), we confirmed that while the gene expression of Cyclin B1 and CDC2 in nasopharynx and pharynx cancer cells treated with CuE decreased, the expression of the GADD45 family (GADD45α, GADD45β, GADD45γ) increased. The results of this study reveal CuE to be an effective drug for head and neck cancer treatment (or adjuvant chemotherapy). The in-depth investigation on the tumor-suppressing mechanisms of CuE and its influence on cell cycles and the protein expression of oncogenes and antioncogenes can provide new direction for the treatment and prevention of head and neck cancers.

並列關鍵字

cucurbitacin E cell cycle autophagy

參考文獻


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