臺灣博碩士論文加值系統

UK-1是一種萃取自鏈黴菌的天然產物，具有抗癌活性。在本篇研究中，我們探討一種新的UK-1衍生物，稱為ACP-93的抗癌活性及機制。人類大腸癌細胞株比其他癌細胞株對ACP-93的細胞毒殺作用較為敏感，將大腸癌RKO細胞以6-24 �嵱 ACP-93處理24小時後，發現隨著藥物處理濃度的提高，ACP-93誘發細胞毒性及細胞週期停滯的現象也隨之上升，ACP-93增加G2/M期並活化CDC2 (threonine-161)蛋白；以CDC2的抑制劑處理，會增加ACP-93所造成的細胞毒性。此外，ACP-93誘發RKO細胞中caspase-3的活化，並造成細胞凋亡。ACP-93增加ERK、p38及JNK蛋白的磷酸化，以ERK的抑制劑PD98059處理，可加強ACP-93所造成的細胞毒性及活化態caspase-3蛋白的含量。相反地，專一性的p38抑制劑SB202190作用時，則可抑制ACP-93所誘發的細胞死亡與caspase-3的活化，然而，以SP600125抑制JNK並不會影響ACP-93所造成的細胞死亡。有趣地，隨著ACP-93濃度的增加，RKO細胞中含氧自由基的產生也隨之增加。若以維生素C處理則可以減少ACP-93所產生的含氧自由基，再者抗氧化劑N-acetylcysteine會顯著地降低ACP-93所造成的細胞毒性。綜合以上結果，我們發現ACP-93是一種有潛力的新合成抗癌藥物，可誘發人類大腸癌細胞週期的停滯及細胞的凋亡，而p38與ERK/CDC2的活化，可扮演正反調控ACP-93所誘發的細胞凋亡，我們也推測含氧自由基的產生參與ACP-93所誘發的細胞凋亡。

UK-1, a natural product isolated from Streptomyces sp, exerts antitumor activity. A new synthetic compound derived from UK-1, ACP-93, was investigated in this study for its anticancer effects and mechanisms. The human colon cancer cell line was more sensitive to the cytotoxicity of ACP-93 than other cancer cell lines. Treatment with 6-24 μM ACP-93 for 24 h induced cytotoxicity and cell cycle arrest via a concentration-dependent manner in RKO colon cancer cells. The fractions of G2/M phases and the protein levels of phospho-CDC2 (threonine-161) were elevated by ACP-93. Treatment with the CDC2 inhibitors increased the cytotoxicity in the ACP-93-treated cells. Moreover, ACP-93 caused the activation of caspase-3 and the induction of apoptosis in RKO cells. The phosphorylation of ERK, p38, and JNK were increased by ACP-93. An ERK inhibitor, PD98059, enhanced the cytotoxicity and increased the active caspase-3 protein levels in the ACP-93-exposed cells. In contrast, a specific p38 inhibitor, SB202190, suppressed the cell death and the activation of caspase-3 in the ACP-93-treated cells. However, the inhibition of JNK by SP600125 did not alter the cell death following treatment with ACP-93. Interestingly, ACP-93 increased the production of reactive oxygen species (ROS) via a concentration-dependent manner in RKO cells. Treatment with vitamin C reduced the ROS generation induced by ACP-93. Furthermore, antioxidant N-acetylcysteine significantly reduced the cytotoxicity in the ACP-93-treated cells. Together, our results suggest that ACP-93 may be a potential anticancer drug, which induces cell cycle arrest and apoptosis in colon cancer cells. The activation of p38 and ERK/CDC2 by ACP-93 exhibits oppositive roles on the regulation of apoptosis. Our data also suggest that the ROS generation by ACP-93 may be involved in the induction of apoptosis.

目錄 頁
目錄........................................................................Ⅰ
圖目錄......................................................................Ⅲ
中英文專有名詞對照表........................................................Ⅳ
中文摘要....................................................................Ⅴ
英文摘要....................................................................Ⅵ
文獻回顧....................................................................1
一、UK-1的衍生物及抗癌活性..............................................1
二、細胞週期............................................................1
三、CDKs/cyclins與細胞週期的調控........................................2
四、細胞凋亡............................................................3
五、caspases與細胞凋亡的調控............................................3
六、有絲抗原蛋白活化激酶之簡介..........................................4
七、有絲抗原蛋白活化激酶與細胞凋亡的調控................................5
八、含氧自由基與細胞凋亡之關係..........................................5
九、研究動機............................................................6
研究材料與方法..............................................................7
一、化學藥品與抗體..........................................................7
二、細胞培養(Cell culture...................................................7
三、細胞毒性分析(Cytotoxicity assay.........................................8
四、細胞凋亡分析(Apoptosis assay............................................8
五、細胞生長分析(Cell growth assay).........................................8
六、細胞週期分析(Cell cycle analysis).......................................9
七、西方墨點法(Western blot)................................................9
八、含氧自由基含量分析(ROS measurement )....................................10
九、間接免疫螢光分析(Indirect immunofluorescence)...........................11
十 共軛焦顯微鏡觀察(Confocal microscopy)....................................12
十一 統計與數據分析(Biostatistics and data analysis)........................12
結果........................................................................13
一、ACP-93誘發不同人類癌細胞之細胞毒性......................................13
二、ACP-93誘發大腸癌細胞生長的抑制及細胞週期的停滯..........................13
三、抑制CDC2活性會促進ACP-93對大腸癌細胞的毒殺作用..........................13
四、ACP-93誘發caspase-3活化及細胞凋亡.......................................14
五、ACP-93誘發有絲抗原活化蛋白激酶的活化....................................14
六、p38激酶與ERK正反調控ACP-93所造成的大腸癌細胞死亡........................15
七、含氧自由基參與ACP-93所誘發大腸癌細胞的死亡..............................15
討論........................................................................17
一、ACP-93誘發人類癌細胞的生長抑制及細胞凋亡................................17
二、CDC2參與ACP-93所誘發細胞死亡............................................17
三、ERK與p38激酶相反調控ACP-93所誘發的細胞凋亡..............................17
四、含氧自由基參與ACP-93所誘發的細胞死亡....................................18
五、總結....................................................................19
參考文獻....................................................................20




圖目錄 頁

圖一、ACP-93對不同人類癌細胞之存活率的影響...................................27
圖二、ACP-93誘發細胞生長抑制................................................ 28
圖三、ACP-93誘發大腸癌細胞G2/M期細胞增加.....................................29
圖四、ACP-93誘發大腸癌細胞中磷酸化CDC2蛋白的表達.............................30
圖五、CDC2抑制劑加強ACP-93所誘發大腸癌細胞的毒性.............................31
圖六、ACP-93誘發大腸癌細胞中caspase-3的活化..................................32
圖七、ACP-93造成細胞凋亡的數目...............................................33
圖八、ACP-93誘發磷酸化ERK、p38及JNK蛋白的表達................................34
圖九、ACP-93誘發磷酸化ERK蛋白增加並聚集於細胞核..............................35
圖十、ACP-93誘發磷酸化p38蛋白增加並聚集於細胞核..............................36
圖十一、ACP-93誘發磷酸化JNK蛋白增加並聚集於細胞核............................37
圖十二、MAP kinases 抑制劑對ACP-93所誘發細胞毒性之影響.......................38
圖十三、PD98059與SB202190對ACP-93造成caspase-3蛋白活化的影響.................39
圖十四、ACP-93誘發RKO大腸癌細胞內含氧自由基量的增加..........................40
圖十五、維生素C降低ACP-93所誘發的含氧自由基的產生............................41
圖十六、N-acetylcysteine降低ACP-93誘發大腸癌細胞的毒性.......................42
圖十七、ACP-93誘發大腸癌細胞凋亡的機轉圖：CDC2、ERK及p38與自由基的角色.......43
附圖一、UK-1化學結構.........................................................44
附圖二、IC50計算方法.........................................................45
附圖三、MAP kinases 抑制劑之化學結構.........................................46

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