王憶卿呂國棟Yi-Ching WangKwok-Tung Lu黃信銘Sin-Ming Huang2019-09-052013-6-242019-09-052011http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN0894430154%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104449　　背景：前人研究指出，多種吲哚類化合物具有抑制細胞分裂的效果，進而導致癌細胞的細胞週期停滯和細胞凋亡。我們實驗室先前的研究證明，3-indole這個新穎的吲哚結構化合物可在30M的濃度下，藉由粒線體途徑（mitochondrial pathway）引起肺癌細胞凋亡，並在動物模式中抑制腫瘤生長。目的：本篇研究探討低濃度3-indole（10M）對癌細胞造成的生長抑制及其分子機轉。另外，我們也發展了另一個新穎的吲哚類化合物—SK228。我們利用細胞及動物模式，探討其對癌細胞生長的影響及其分子機轉。結果：在以10M 3-indole處理A549、CL1-1及H1437肺癌細胞株之後，發現癌細胞生長被抑制，並且造成細胞週期停滯於G1期。3-indole引起的DNA損傷由Comet assay所驗證，並且數種DNA損傷反應蛋白質及G1期調控蛋白質（例如RB、p53、p21和 SMAD3）在3-indole處理後表現量增加。我們進一步發現，3-indole也會引起DNA損傷反應路徑—ATM/ATR路徑的活化；另外，活性氧自由基（reactive oxygen species, ROS）的抑制劑rotenone能降低3-indole引起的DNA損傷和ATM/ATR反應路徑的活化。這些結果指出，ROS在3-indole引起的癌細胞生長抑制中，扮演重要的角色；動物異種移植（xenograft）實驗進一步證明，3-indole在細胞模式中活化的反應路徑，亦可見於動物模式中。在SK228的研究中，我們發現該化合物可有效的抑制肺癌及食道癌細胞生長，而對於正常肺纖維母細胞則無明顯影響。在以SK228處理過的細胞中，觀察到代表細胞凋亡的「細胞膜內膜外翻」的現象，指出SK228會引起細胞凋亡。我們進一步驗證了，SK228藉由與DNA的結合、鑲嵌及產生ROS而造成DNA的結構改變及損傷。SK228的處理會促進cytochrome c從粒線體釋放至細胞質中，以及caspase-3 和 caspase-9的活化，但不影響caspase-8的活性，並且這些反應可被ROS的抑制劑rotenone所抑制。另外，BCL-2家族的蛋白質表現量及粒線體外膜的完整性，亦受到SK228的影響。我們更進一步發現，SK228藉由降低FAK/paxillin路徑及RhoA的活性而抑制癌細胞的轉移能力。另外，動物實驗也證明SK228可有效抑制腫瘤細胞的生長，並且沒有引起明顯的體重變化或血液學、生化學上的明顯傷害。而且，藉由TUNEL assay和免疫組織化學染色證明，SK228可在動物模式中誘發癌細胞的細胞凋亡，進而抑制腫瘤生長。結論：本實驗證明，低濃度3-indole會藉由產生ROS而造成DNA損傷，並引起ATM/ATR路徑和TGF-β/SMAD路徑的活化，進而使細胞產生細胞週期G1期停滯現象。另外，SK228會藉由造成DNA損傷及ROS的產生而誘使癌細胞經由粒線體路徑進行細胞凋亡，並且也證明SK228可以在低濃度下有效抑制癌細胞的轉移能力。Background: In our previous study, we demonstrated that 30μM of 3-indole, a new generated indole compound, induces lung cancer cells apoptosis through intrinsic mitochondrial pathway and inhibits tumor growth in xenograft models. Purpose: This study aims to verify the effects and mechanism of low concentration (10μM) of 3-indole treatment on cancer cell growth. In addition, we developed a novel indole compound 3,3',3'',3'''- (1,4-phenylenebis(methanetriyl))tetrakis (1H-indol-5-ol), named SK228. Its effects and mechanism on anticancer growth and migration were examined in cell and animal models. Results: Cell growth inhibition and cell cycle G1 arrest were observed in A549, CL1-1, and H1437 lung cancer cell lines treated with 10μM 3-indole. Several DNA damage responsive proteins and G1 regulation proteins such as RB, p53, p21, and SMAD3 were also induced. DNA damage was confirmed by Comet assay. We further found that DNA damage response ATM/ATR pathway proteins, such as ATM, CHK1, and CHK2 can be induced in 3-indole treated A549 cells. Interestingly, reactive oxygen species (ROS) inhibitor rotenone reduced the DNA damage effect, abated the level of ATM/ATR pathway proteins, and decreased the level of G1 cell cycle arresting proteins induced by 3-indole. The altered expression of ATM/ATR pathway proteins were confirmed in xenograft models. These results indicated the crucial role of ROS in cancer call growth inhibition by 3-indole. In the study of SK228, we found its significant cell growth inhibition efficiency on lung cancer and esophageal cancer cell lines, without affecting the growth of normal lung cell line IMR90. After treating with SK228, induction of phosphatidylserine staining with an increase of sub-G1 phase were observed, indicating that SK228 induced cell apoptosis. SK228 induced DNA damage by Comet assay, and SK228-induced damage resulted from DNA minor groove binding, DNA intercalating, and ROS production. SK228 treatment increased the release of cytochrome c into cytosol along with the increased activity of caspase-3 and -9 without affecting caspase-8, whereas these effects were attenuated by ROS inhibitor. In addition, the expression levels of BCL-2 family regulators of mitochondrial outer membrane permeablization were also affected. These results indicated that SK228 induced apoptosis mainly through intrinsic/mitochondrial pathway. Moreover, low-dose SK228 significantly reduced the invasion of cancer cells. The FAK/paxillin signaling pathway proteins and active form of RhoA were decreased. The F-actin cytoskeleton was also disrupted. Animal study showed that SK228 remarkably reduced tumor size in nude mice bearing human A549 lung carcinoma tumor xenograft without significant side effects. In addition, TUNEL assay and immunohistochemistry of cleaved caspase 3 in tumors from SK228-treated animals confirmed that SK228 inhibited cancer cell growth by initiating apoptosis. Conclusions: This study showed that low concentration 3-indole causes DNA damage by ROS and triggers ATM/ATR-CHK1/CHK2 and TGF-β/SMAD signaling pathway to arrest lung cancer cells in G1 phase. In addition, we demonstrate SK228 caused DNA damage and ROS production leading to cancer cells apoptosis through mitochondria-mediated pathway in cell lines and animal models and inhibited cancer cell invasion via FAK/paxillin disruption at non-cytotoxic doses.吲哚DNA損傷活性氧化物細胞凋亡indoleDNA damagereactive oxygen speciesapoptosis