探討葫蘆苦素E (CuE) 誘發經由GADD45β調控CDC2/Cyclin B複合物解離導致人類膠質母細胞瘤細胞株(GBM) 細胞週期有絲分裂期停滯

Cucurbitacin E (CuE) induce GADD45β mediated prolonged delay in mitosis by CDC2/Cyclin B complex disassociated in human brain malignant (GBM) cell lines

洪琳捷(Lin-Chieh Hung)

指導教授 ： 徐怡強

長榮大學/健康科學學院/醫學研究所/碩士(2016年)

摘要

根據衛生福利部國民健康署最新公佈的國人十大死因中，惡性腫瘤自71年起已連續33年高居國人死因之首位。Glioblastoma malignant (GBM) 為一極度惡性的神經膠母質細胞瘤，佔所有腦瘤發生率一半以上且具有高死亡率、高發生率及抗藥性高。常見的癌症治療方法為外科手術、化學藥物治療、標靶治療等，皆會對正常細胞產生不良副作用。因此近年來有許多研究以中草藥或天然萃取物當作輔助性藥物或是副作用較低的抗癌藥物來進行研發。
葫蘆苦素 E (Cucurbitacin E; CuE ) 是四環三萜類化合物 (tetracyclic triterpenes) ，由葫蘆科一年生蔓性草本植物甜瓜 (Cucurmic melo L) 的瓜蒂與果柄萃取而來，具有抗發炎、抗細胞毒性及抑制細胞生長等生物活性。本碩士論文主要探討葫蘆苦素 E對於人類膠質母細胞瘤細胞株 (GBM 8401、U-87-MG) 抗腫瘤生長效果與誘發的分子機轉。
透過體外實驗來探討葫蘆苦素 E的抗腫瘤效果，利用人類膠質母細胞瘤細胞株進行分析，以 MTT assay 分析細胞的存活率，結果顯示葫蘆苦素 E 能顯著抑制癌細胞生長，且有效抑制人類膠質母細胞瘤細胞的生長並造成細胞週期 G2/M 期的停滯現象，再藉由 Real-time PCR 分析細胞週期基因表現量，如： CDC2 、 Cyclin B 、 MAPK 家族等，再進一步運用西方墨點法來檢測細胞週期 G2/M 期的調控相關蛋白質含量。
除了探討葫蘆苦素 E 對腦癌細胞之抑癌作用機轉與細胞週期，在本論文更發現 GADD45β 調控 CDC2/Cyclin B 複合物，誘導細胞週期停滯，因此利用 GADD45/MKK7 抑制劑 (DTP3) 降低 GADD45β 之活性及小干擾 RNA 抑制其基因表現，可反向證明葫蘆苦素 E 可藉由 GADD45β 降低 CDC2/Cyclin B 複合物鍵結力所導致的細胞週期 G2/M 停滯。本研究發現葫蘆素 E 可作為有效的腦癌治療或輔助化學治療用藥物，期許在未來能夠提供腦癌治療及預防的最佳方案。

關鍵字

葫蘆苦素E ； 細胞週期G2/M 停滯 ； GADD45β

並列摘要

The Ministry of Health and Welfare, National Health Department, has recently identified malignant tumors as the leading cause of death in Taiwan. Glioblastoma multiforme (GBM) is the most common malignant brain cancer, which accounts for more than half of all brain tumors. GBM has a high mortality rate and is recurrent and resistant to chemotherapy. Common cancer therapies, including surgery, chemotherapy, and targeted therapy, induce many side effects on normal cells and tissues. Extracts of herbal or natural products have proved to be complementary medicine with fewer side effects than chemotherapy drugs. Cucurbitacin E (CuE), a tetracyclic triterpene extracted from the climbing stem of melon, is shown to have anti-inflammatory and anti-cytotoxic responses and inhibitory effects on cancer cell growth.
In this study, we investigated the anti-tumor effects of CuE in human brain cancer cell lines (GBM 8401, U87-MG). Cell survival was analyzed using the MTT assay. Our results showed that the GBM cell lines treated with CuE had significantly inhibited cell growth. CuE treatment was found to induced cell cycle G2/M phase arrest but not apoptosis or necrosis in the human brain cancer cell lines.
The expression profile of G2 /M phase-associated proteins, such as CDC2 and cyclin B, and p38/JNK pathway regulation was analyzed by Western blotting. Our findings indicated that the expression of these proteins is downregulated in the GBM cell lines by CuE treatment. CuE may be an effective candidate for brain cancer treatment or adjuvant chemotherapy. Further, it may provide a new direction in brain cancer prevention and treatment.

並列關鍵字

Cucurbitacin E ； Cell cycle G2/M arrest ； GADD45β

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