臺灣博碩士論文加值系統

COQM 是一種以查耳酮(chalcone)及喹啉(quinolone)合成的新化合物，且查耳酮和喹啉都已被證實具有抗癌和抗發炎的能力。然而，目前對於 COQM 實際用於乳癌治療的成效尚不清楚。本文旨在探討 COQM 對於三陰性乳癌細胞(HCC1937)及非三陰性乳癌細胞(SKBR-3)的效果，以及藉由確認其是否會傷害正常乳房細胞(H184B5F5/M10, M10)來探討 COQM 作為藥品的安全性。經由實驗可知，COQM 顯著抑制了三陰性乳癌細胞(HCC1937)及非三陰性乳癌細胞
(SKBR-3)之生長、促進細胞停滯於細胞週期中的 subG1 期，並藉由增加 Caspase 3、Caspase 8 和 Caspase 9 之表現來大幅促進細胞凋亡。然而，上述的效果在正常乳房細胞(M10)中都遠低於乳癌細胞。不僅如此，這些效果都能被 Nacetylcysteine (NAC)減弱，這也代表 COQM 經由調控氧化壓力來促進細胞凋亡。因此本篇研究也藉由流式細胞儀確認COQM 對於氧化壓力的影響，並發現COQM 能增加細胞內的活性氧物質(reactive oxygen species)及粒線體超氧化物(mitochondrial superoxide)堆積，還能使粒線體膜電位及穀胱甘肽(glutathione)含量顯著下降，並促進 DNA 雙股和單股斷裂。上述效果在乳癌細胞中遠高於正常細胞，且同樣能被 NAC 減弱，顯示 COQM 具有選擇性毒殺之特性。因此可知，COQM 能藉由調控氧化壓力來達成在乳癌細胞中的抗癌效果，且對於正常細胞的傷害較低，在未來對乳癌的治療上將會是很好的研究目標。

COQM is a newly developed hybrid compound of chalcone and quinolone. Both are reported to have anticancer and anti-inflammatory abilities, but the effect of COQM on breast cancer cells and drug safety was poorly understood. This study aims to assess the anticancer ability and mechanism of COQM against triple-negative breast cancer (TNBC; HCC1937) and non-TNBC (SKBR-3) cells compared to normal breast cells (H184B5F5/M10 (M10)). COQM exhibits more antiproliferation, subG1 accumulation,apoptotic phenotype (annexin V), and activates apoptotic signaling such as caspases 3, 8, and 9 in breast cancer than normal cells. These effects are attenuated by Nacetylcysteine (NAC) pretreatment, suggesting oxidative stress mediates these antibreast cancer mechanisms. The COQM-induced oxidative stress responses are further validated by flow cytometry, showing higher generation of reactive oxygen species and mitochondrial superoxide and lower mitochondrial membrane potential and glutathione in breast cancer than normal cells, attenuated by NAC. Similarly, COQM exhibits oxidative stress-dependent DNA damages for γH2AX and 8-hydroxy-2-deoxyguanosine in a greater extent of breast cancer than normal cells, confirmed by NAC. Consequently, COQM has anticancer effects and oxidative stress-mediated mechanisms in breast cancer cells and exhibits low impact on normal cell.

目錄
中文摘要.......................................................................................................................1
英文摘要.......................................................................................................................2
1. 背景介紹.................................................................................................................3
1.1 乳癌分類及現今治療方法 ..............................................................................3
1.2 查耳酮及喹啉之抗癌特性 ..............................................................................3
1.3 化療藥物泰嘉錠之療效..................................................................................4
1.4 COQM 簡介....................................................................................................4
2. 研究動機與目的.....................................................................................................5
2.1 假說 .................................................................................................................5
2.2 實驗流程圖......................................................................................................6
3. 實驗材料與方法.....................................................................................................7
3.1 細胞培養 .........................................................................................................7
3.2 藥物製備 .......................................................................................................14
3.3 細胞存活率分析(ATP assay)........................................................................16
3.4 細胞週期分析(Cell cycle analysis)...............................................................19
3.5 細胞凋亡分析(Apoptosis assay)...................................................................22
3.6 Caspase 3/7 活性分析(Caspase 3/7 analysis)...............................................25
3.7 Caspase 3/8/9 含量測定(Caspase 3/8/9 activity assay)................................27
3.8 ROS 含量測定(ROS assay)..........................................................................30
3.9 粒線體內之超氧化物測定(MitoSOX assay)................................................33
3.10 粒線體膜電位檢測 (MMP assay)................................................................36
3.11 GSH 含量測定 (GSH assay)........................................................................39
3.12 DNA 雙股損傷測定(γH2AX assay) .............................................................42
3.13 氧化性 DNA 損傷測定(8-OHdG assay) ......................................................47
4. 實驗結果說明.......................................................................................................50
4.1 COQM 透過調控 ROS 來抑制細胞增生.....................................................50
4.2 COQM 改變乳癌細胞之細胞週期...............................................................50
4.3 COQM 選擇性增加乳癌細胞之細胞凋亡率...............................................50
4.4 COQM 選擇性增加乳癌細胞之 caspase3/7 活性 .......................................51
4.5 COQM 選擇性促進 caspase 8 和 caspase 9 之活化....................................51
4.6 COQM 選擇性增加乳癌細胞中之 ROS 堆積量.........................................52
4.7 COQM 選擇性增加乳癌細胞中之 MitoSOX 含量.....................................52
4.8 COQM 選擇性降低乳癌細胞之粒線體膜電位 ...........................................53
4.9 COQM 選擇性減少乳癌細胞之 GSH 含量.................................................53
4.10 COQM 選擇性提升乳癌細胞中 γH2AX 之生成量.....................................54
4.11 COQM 選擇性促進 8-OHdG 之含量提升 ..................................................54
5. 討論 ......................................................................................................................55
5.1 查耳酮及喹啉之抗乳癌療效 ........................................................................55
5.2 COQM 透過增加氧化壓力抑制乳癌細胞之生存率 ...................................56
5.3 COQM 大幅提升乳癌細胞的 subG1 期......................................................56
5.4 COQM 藉由調控氧化壓力促進乳癌細胞凋亡 ...........................................57
5.5 COQM 增加乳癌細胞之氧化壓力並損壞 DNA..........................................57
5.6 比較 COQM 與臨床化療藥物 ciaplatin 之效果 .........................................58
6. 結論 ......................................................................................................................59
7. 實驗結果圖...........................................................................................................60
7.1 細胞存活率分析(ATP assay)........................................................................60
7.2 細胞週期分析(Cell cycle analysis)...............................................................62
7.3 細胞凋亡分析(Apoptosis assay)...................................................................63
7.4 Caspase 3 活性分析(Caspase 3 analysis).....................................................65
7.5 Caspase 8/9 活性測定(Caspase 8/9 activity assay)......................................67
7.6 ROS 含量測定(ROS assay)..........................................................................69
7.7 粒線體內之超氧化物測定(MitoSOX assay)................................................71
7.8 粒線體膜電位測定(MMP assay)..................................................................73
7.9 GSH 含量測定 (GSH assay)........................................................................75
7.10 DNA 雙股損傷測定(γH2AX assay) .............................................................77
7.11 氧化性 DNA 損傷測定(8-OHdG assay) ......................................................79
8. 參考文獻...............................................................................................................81

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