肺癌是常見之惡性腫瘤，也是癌症致死的主因。近年來國人肺癌罹患日漸增加，在女性癌症所佔之比率尤有居高不下之趨勢。本校郭盛助教授實驗室所合成之3΄-Methoxy-6 pyrrolinyl-2-phenyl-4- quinolone (K3) 已在離體實驗 (in vitro) 證明具有抑制tubulin 聚合之能力，因此，我們用常見之肺鱗狀上皮細胞癌 (CH27細胞) 測試K3之抗癌活性及機制。分析K3處理3天後之存活率發現K3對CH27肺癌細胞具強毒殺性，其IC50濃度約10 nM 。K3對HL-60之IC50濃度亦遠小於8 nM以下。以1 μM K3分別處理CH27肺癌細胞1天之細胞存活率約55.6%，第2天約16.8%，第三天時細胞存活率只剩0.098%。用流式細胞儀分析經1 μM K3或1 μM Colchicine處理後之肺癌細胞核DNA含量，結果顯示隨著時間增加G2/M期之細胞比率逐漸增加；K3處理24小時後約 55.6% 的細胞停滯於G2 /M期，過了24小時後，細胞漸走向凋亡。類似的變化亦在另一肺癌細胞A549及血癌細胞HL60及U937觀察到。觀察三天細胞形態變化， K3處理時間越久，貼著細胞數就越少，圓狀化細胞愈多。DAPI及TUNEL染色結果顯示，K3處理72小時之細胞呈現核斷裂的現象。以免疫螢光染色法分析此時細胞內之Caspase 3及微管之構造，結果顯示Caspase-3顯著增加，且K3 會抑制微管 (microtubule) 的聚合。洋菜膠電泳分析發現1 μM K3處理48小時後，核內DNA呈梯度片段。由西方墨點法分析結果可發現1 μM K3 使CH27細胞之Bcl-2蛋白質量微量減少，而Bad則明顯增加， Cyclin B明顯增加、Cyclin A減少、CDK1亦減少。同時我們也發現K3 使粒線體膜電位下降及氧化自由基 (ROS) 上升。由以上結果顯示，K3 抑制人類肺癌細胞的增殖作用與誘導細胞停滯於G2 /M期及細胞凋亡有關。; Cancer is the leading cause of death in our country. Amongst all kinds of cancers, lung cancer is currently the one with the highest mortality. Therefore, it is important to explore the new anti-cancer drugs and improve therapeutic strategies for lung cancer treatment. In this thesis, we studied one of the newly synthesized 3΄,6,7-substituted 2-phenyl-4-quinolones compounds from Dr. Kuo Sheng-Chu that has been reported to inhibit tubulin polymerization and be cytotoxic to cancer cell lines. Among them, compound 3΄-Methoxy-6 pyrrolinyl- 2-phenyl-4-quinolone (K3) was found to be the most potent one. Human lung cancer cell CH27 was used to investigate anti-cancer effects of K3 on cell proliferation, cell cycle progression, and induction of apoptosis. This current study found that K3 was cytotoxic to human lung cancer cell with IC50 at 10 nM approximately. Cell cycle analysis demonstrated that 24 hours-K3-treated CH27 were mainly arrested at the G2/M phase. Afterward, sub-G1 population gradually increased with time. Within 72 hours of treatment, K3 induced a time dependent generation of typical DNA-laddering and apoptosis- specific morphological changes by DNA gel electrophoresis, Giemsa and DAPI staining, and TUNEL assay. Furthermore, immuno-fluorescence staining demonstrated the significant increase of Caspase-3 and the inhibition of tubulin polymerization after K3 treatment. Western blotting revealed that K3 induced the increase of Cyclin B after 24 hours of treatment. However protein levels of Cyclin A and CDK1 were decreased. The expression of anti-apoptotic Bcl-2 was decreased but the pro-apoptotic Bad was increased. Analysis from flow cytometry indicated that the mitochondria membrane potential was decreased and the amount of ROS (reactive-oxygen species) was increased. These results indicated that the ability of K3 to inhibit cancer cell is mainly mediated by the induction of G2/M arrest and apoptosis.
