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研究生:王建穎
研究生(外文):Jian-Ying Wang
論文名稱:探討兩種新穎性Nilotinib衍生物對人類大腸癌細胞幹性的影響
論文名稱(外文):Assessing the effects of two novel nilotinib derivatives in the stemness of human colon cancer cells
指導教授:蘇瑀蘇瑀引用關係
指導教授(外文):Yeu Su
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:30
中文關鍵詞:大腸癌幹細胞nilotinib衍生物單磷酸腺苷活化蛋白激酶
外文關鍵詞:colon cancer stem cellnilotinib derivativesAMPK
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研究顯示大腸癌症幹細胞(colorectal cancer stem cell, CRCSCs)與癌症的啟始、復發、轉移和抗藥性有密切的關係,導致癌症患者預後不佳。而單磷酸腺苷活化蛋白激酶(AMP-activated protein kinase,以下簡稱AMPK)為調控能量衡定的重要酵素,其主要功能為協調代謝路徑、平衡營養供給和能量需求。在內生性AMPK活性較低的癌細胞中,細胞代謝較為迅速,有效的運用葡萄糖,使癌細胞快速增生。Nilotinib為治療慢性骨髓性白血病(chronic myeloid leukemia, CML)的第二線用藥,根據先前的研究發現,該藥物能透過活化AMPK誘導細胞自噬而抑制肝癌細胞生長,因此在本研究中我分析由本校藥學系蘇溶真老師提供,具有AMPK活化作用的nilotinib衍生物SCT-1015及SCT-1051,兩者對HCT-116及HT-29人類大腸癌細胞幹性的影響,以及探討其可能的作用機制。
首先以MTT分析法檢測SCT-1015及SCT-1051對HCT-116和HT-29細胞的毒性,發現其IC50分別為2.7和5.6 μM(SCT-1015)以及20.8和9.9 μM(SCT-1051)。接著以SCT-1015(2 和6 μM)和SCT-1051(1和4 μM)分別處理HCT-116和HT-29細胞不同時間後,分析AMPK被活化的狀況,發現SCT-1015和SCT-1051分別在作用8~12及24小時後可以活化HCT-116細胞內的AMPK,但無法活化HT-29細胞內的酵素。後續發現SCT-1015之抑制HCT-116及HT-29細胞形成腫瘤球體的IC50分別為1.72和0.78 μM,而SCT-1051則只能抑制HT-29細胞形成腫瘤球體,其IC50為0.74 μM。接著也發現SCT-1015可引發兩種細胞已形成之腫瘤球體的萎縮,IC50分別為1.86及4 μM;SCT-1051則無法造成腫瘤球體的萎縮,進而根據細胞在軟洋菜膠內形成聚落試驗的結果,發現兩種藥物對HCT-116細胞均無明顯的抑制作用,而SCT-1015抑制HT-29細胞非貼覆性生長的IC50為0.85 μM。根據RT-qPCR的分析結果,發現SCT-1015處理會增加HCT-116和HT-29細胞內的LGR5 mRNA表現量,兩種藥物皆能降低HCT-116細胞中ALDH1A1和BMI1的 mRNA表現量,但LGR5和ASCL2表現只受SCT-1051抑制。而在幹性蛋白表現部分,發現經SCT-1015處理後HCT-116細胞Bmi1表現量下降,而HT-29細胞則是ALDH1A1和Ascl2表現被抑制。目前實驗的結果顯示SCT-1015和SCT1051雖然能抑制HCT-116和HT-29人類大腸癌細胞的自我更新,非貼覆性生長以及降低特定癌細胞幹性基因和蛋白的表現,但仍無法確定這些幹性抑制作用是否與活化AMPK有關,目前已在利用CRISPR-Cas9系統製備AMPKα2基因敲除的HCT-116細胞株來回答上述的問題。
Ample evidence has shown that colorectal cancer stem cells (CRCSCs) are responsible for the initiation, metastasis, recurrence, and drug resistance of this malignancy, leading to a poor prognosis in CRC patients. AMP-activated protein kinase (AMPK) is an important enzyme regulating energy balance whose main function is to coordinate metabolic pathways, hence balancing nutrient supply and energy demand. In cancer cells with low endogenous AMPK activity which proliferate rapidly because they metabolize more quickly and utilize glucose more effectively. Nilotinib, a second-line drug for treating chronic myeloid leukemia (CML), has been shown to inhibit the growth of human hepatoma cells by inducing autophagy via activating AMPK.
In this study, we analyzed the effects of two novel nilotinib derivatives, SCT-1015 and SCT-1051 provided by Dr. Su J.C. of the Department of Pharmacy of our University, on the stemness of HCT-116 and HT-29 human CRC cells as well as dissected their possible mechanisms. MTT assays were first used to examine the respective cytotoxic effects of SCT-1015 and SCT-1051 on HCT-116 and HT-29 cells and we found that the IC50 values of the former and the latter in HCT-116 and HT-29 cells were 2.7 and 5.6 M and 20.8 and 9.9 M, respectively. HCT-116 and HT-29 cells were then treated with SCT-1015 (2 & 6 M) and SCT-1051 (1 & 4 M) for varying lengths of time before the levels of active AMPK were analyzed. Interestingly, SCT-1015 and SCT-1051 could activate AMPK in HCT-116 cells after 2-12-hr and 24-hr treatment, respectively. But neither compound could activate this enzymes in HT-29 cells. Subsequently, we found that SCT-1015 inhibited the formation of tumor spheres of HCT-116 and HT-29 cells with IC50 values of 1.72 and 0.78 M, respectively, whereas SCT-1051 only inhibited that of HT-29 cells with an IC50 of 0.74 M. Surprisingly, we observed that only SCT-1015 could induce shrinkage of tumor spheres formed by both CRC lines with IC50 values of 1.86 and 4 M, respectively. Moreover, results of soft agar colony forming assay showed that SCT-1015 suppressed anchorage-independent growth of HT-29 cells with IC50 values of 0.85 M. According to the results of RT-qPCR analysis, SCT-1015 treatment increased the levels of Lgr5 mRNA in HCT-116 and HT-29 cells. Both drugs could reduce the mRNA levels of ALDH1 and Bmi1 in HCT-116 cells, but those of Ascl2 were only decreased by SCT-1051. However, results of western blotting showed that SCT-1015 markedly reduced protein levels of Bmi1 and ALDH1A1 in HCT-116 and HT-29 cells, respectively.
Together, although SCT-1015 and SCT1051 seem to inhibit certain stemness properties of HCT-116 and HT-29 human CRC cells, whether these effects are mediated by AMPK activation is still unknown. Hence, our current work focuses on establishing the AMPK2 knockout HCT-116 cells using the CRISPR-Cas9 system and will use them to answer the above question.
目錄
誌謝.............I
摘要.............II
ABSTRACT.........III
目錄..............IV
圖次目錄...........V
緒論 1
研究動機 5
材料與方法 6
實驗結果 12
討論 15
參考文獻 17
APPENDIX 1. 30
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