膠質瘤(Glioma)是成人之中最為惡性的腦腫瘤，一般治療方式為合併手術、放射線治療以及化療，但是患者的存活率仍然無顯著提升。秋水仙素(Colchicine)為微管蛋白的抑制物，通常被用來治療痛風，也被做為抗癌藥物的使用。過去文獻指出利用秋水仙素可用來治療乳腺癌及大腸癌，而使用秋水仙素治療膠質瘤則較少被研究，並且秋水仙素必須使用至micromole濃度才能達到抑制效果，如此高濃度對正常細胞造成毒害。本研究想探討新型秋水仙素衍生物AD1是否能夠以低濃度就能有效抑制惡性膠質瘤。我們使用兩株第四級的惡性膠質瘤細胞株:U373MG及U87MG探討秋水仙素衍生物AD1的作用。α-tubulin免疫螢光染色結果顯示AD1濃度10 nM 處理U87MG 24小時已造成明顯細胞損傷，但是10 nM並未造成明顯 U373MG細胞形態改變，AD1濃度提高至50 nM才造成明顯U373MG細胞結構上的變化。利用Trypan blue staining結果顯示，相對於控制組，AD1 濃度在10 nM 和50 nM 減少U87 MG 和U373MG細胞數目，且延長處理時間達72小時，細胞數目更明顯被抑制。更進一步利用流式細胞儀分析24小時處理AD1，U87MG 和U373MG細胞內 ROS量有明顯上升。免疫螢光染色以及西方點墨法顯示處理AD1後會使細胞自噬相關蛋白LC3I轉換成LC3II，代表細胞的自噬小體(autophagosome)增加。Acridin orange染色合併流式細胞儀分析結果說明處理AD1可以誘導細胞自噬。然而，前處理PI3K抑制劑Wortmannin以及LY294002，導致更低濃度(5 nM)的AD1造成此兩種膠質瘤細胞株細胞損傷。依目前研究結果，我們結論處理秋水仙素衍生物AD1在低濃度(10 nM-50 nM)可以導致惡性膠質瘤細胞氧化壓力上升造成細胞損傷並伴隨AD1細胞自噬的現象。

Glioblastoma is the most malignant brain tumor in adults. Conventional treatments include a combination of surgery, radiotherapy, and chemotherapy. However, the poor survival rate remains. Colchicine, one of the microtubule inhibitor, is a common therapeutical agent for gout, and also used as an antitumor drug with side effects through disrupt the microtubule structure. Accordingly, the development of its derivatives with low dose efficacy and less side effect could bring impact on anti-tumor treatment. In this study, we present the effect of a colchicine derivative so named as AD1 on U373MG and U87MG, two grade IV human glioblastoma cell lines. Through immunostaining of α-tubulin, we found that the treatment of U87MG cells with AD1 for 24 h at 10 nM, led to severe cell damage. The same observation was found in U373MG cells with AD1 at 50 nM. Trypan blue cell counting analysis showed that AD1 caused the cell death of U373MG and U87MG cells in a dose-dependent manner and AD1 repressed the cell viability of U373MG and U87MG cells for 72h. We also found that increased levels of ROS in U373MG and U87MG cells treated with AD1. Further experiments revealed that the application of AD1 increased the conversion of LC3-I to LC3-II in U373MG and U87MG cells, indicating that AD1 may induce the autophagy of the two human glioblastoma cell lines. Acridine orange staining for autophagic vesicles analyzed by flow cytometry showed that treatment with AD1 induced the autophagy in U373MG and U87MG cells. However, pretreatment with PI3K inhibitor, Wortmannin and LY294002, resulted in cell damage with AD1 at 5 nM in U373MG and U87MG cells. Taken together, we conclude that treatment with AD1 causes the glioblastoma cells death along with the occurrence of autophagic process.

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