以肺癌細胞株與動物模式探討新穎的吲哚結構合成化合物. 1,1,3-tri(3-indolyl)cyclohexane 抑制腫瘤細胞生長機制

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1 國立臺灣師範大學生命科學系博士論文 以肺癌細胞株與動物模式探討新穎的吲哚結構合成化合物 1,1,3-tri(3-indolyl)cyclohexane 抑制腫瘤細胞生長機制 A novel two-step synthetic indole compound 1,1,3-tri(3-indolyl)cyclohexane inhibits cancer cell growth in lung cancer cells and xenograft models 研究生 : 李慶孝 Ching-Hsiao Lee 指導教授 : 王憶卿博士 李桂楨博士 Dr. Yi-Ching Wang, Dr. Guey-Jen Lee-Chen 中華民國九十七年七月

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4 謝誌 這些年來, 畢業是永不放棄的念頭, 終於, 這一天到來 感謝指導教授王憶卿老師在我博士班生涯轉折點出現, 並一直給予最大的幫助, 使得實驗及論文能順利完成, 感激之情, 無法言喻, 謹致上我最深的謝意 研究期間承蒙國衛院癌研所張俊彥醫師 臺中榮總教研部徐士蘭博士 中山醫學大學生化所謝易修教授和臺北榮總劉宗榮老師在實驗瓶頸時, 提供實驗方向 觀念導正 儀器使用的協助, 亦致上衷心之敬謝 同時謝謝在各實驗室的朋友 : 國衛院國順 政智 永齡 靜芬 靜娟, 臺中榮總鄭旗志學長實驗技術的悉心教導, 和妳 ( 你 ) 們合作的這段日子很快樂 竹南動物科技研究所王耀宏學長 台灣大學動物科學技術學系吳信志教授 高師大生物科技系梁世雄老師在困難時的資助 師大李桂楨老師 蘇銘燦老師給予的學業及生活上的協助 此外, 臺北榮總許翰水醫師及其助理宜君在實驗及精神上各方面的幫忙在此亦一併致謝 共同渡過論文過程中無數個日夜及重複實驗的夥伴建智 韋倫 子憲 哲維 金祝 若嘉 若凱 秋逸 一泓 芳宜 信銘 昆學 世華 千惠 豐任 本翰 家揚 偉伶 素芬 一麟 翊萱 孝文 貝君 侑庭 與關心幫忙的師大的所有朋友瑞宏 政光 玄原 國保 承岳 葦苓 士寰 舒如 伯寬 李姐 柏安 欣杰 芳足 添順, 因為有大家的陪伴, 讓我孤寂的生活增趣不少, 真心感謝大家

5 此外, 仁德醫專的同事意文 羅文星老師 小芳 子華 ( 現為新 竹教育大學助理教授 ) 欣凱 郭蕙嵐老師 賴志河學長 ( 現為中國醫藥 大學助理教授 ) 幸樺 秀梅 ( 現為中國醫藥大學助理教授 ) 嘉杏 冠 豪 雪玉, 若沒有妳 ( 你 ) 們的協助, 無法在無後顧之憂的情況下 安心進修, 尤其侯建維學長不時的叮嚀身體健康, 在此謝謝大家, 並致上最誠摯的感激 時光荏苒, 七年波折的博士班生活, 同時也開始了另一段人生 : 婚姻和女兒 弟弟出生 對於母親及家人多年來, 無怨無悔的容忍與等待, 在此深表感恩的心意 此外, 高興有女兒妞妞 ( 沅恩 ) 以及弟弟 ( 沅宸 ) 的加入, 每一次疲憊不堪的辛苦, 都因兩個寶貝可愛的臉龐而煙消雲散 謹以此文獻給天人永隔卻與我常在的父親及所有陪我渡過挫折與快樂的家人 師長與朋友們 慶孝謹誌 國立台灣師範大學生命科學系 中華民國 97 年 7 月

6 CONTENT 以肺癌細胞株與動物模式探討新穎的吲哚結構合成化合物 1,1,3-tri(3-indolyl)cyclohexane 抑制腫瘤細胞生長機制 A novel two-step synthetic indole compound 1,1,3-tri(3-indolyl)cyclohexane inhibits cancer cell growth in lung cancer cells and xenograft models Chinese Abstract English Abstract Introduction I. Outline of lung cancer II. Overview of cell cycle Cell cycle Cell cycle checkpoint Cyclin-dependent kinases inhibitors (CKIs) III. Overview of apoptosis i

7 1. Apoptosis Pathways of Apoptosis Caspases (cysteine-dependent aspartate-specific proteases) Bcl-2 family Ⅳ. Reactive oxygen species (ROS) V. The mitogen-activated protein kinase (MAPK) family Ⅵ. Compounds with an indole structure Microtubule structure and function Anti-microtubule drugs Ⅶ. 1,1,3-tri(3-indolyl)cyclohexane (3-indole) Materials and Methods Ⅰ. 1,1,3-tri(3-indolyl)cyclohexane (3-indole) Ⅱ. Cell Culture Ⅲ. Cell Proliferation Assay Ⅳ. Analysis of Cell Cycle Distribution V. Determination of the Apoptotic DNA Ladder Ⅵ. Evaluation of the Mitochondrial Transmembrane Potential Ⅶ. Western Blot Analysis Ⅷ. Determination of Caspase Activity IX. Immunocytochemistry X. Determination of Intracellular Reactive Oxygen Species XI. Pulsed-Field Gel Electrophoresis XII. cdna Microarray Analysis ii

8 XIII. Subcutaneous Implantation of Cancer Cells in Animals and Monitoring of in Vivo Anti-tumoral Activity afters Drug Treatment Results I. 3-indole Apparently Inhibited Growth at Low Concentration and Promoted Cell Death at High Concentration in Various Human Lung Cancer Cells II. 3-indole Induced Cell Cycle Arrest and Apoptosis in Various Human NSCLC Cells III. Activation of the p53/p21 Pathway Is Required for the Induction of Cell Cycle Arrest in 3-indole IV.3-indole Induced Apoptosis Through the Activation of the Intrinsic Mitochondrial Pathway V.3-indole Induced Cell Cycle Arrest and Apoptosis by Reactive Oxygen Species Production and DNA Double-Strand Breaks in A549 or H1299 Cells VI.cDNA Microarray Analysis to Search For Differential Expressed Genes After 3-indole Treatment VII.Activation of the JNK Signaling Pathways Is Required for the Induction of Apoptosis in 3-indole Treated A549 Cells VIII.3-indole Effectively Inhibited the Growth of Human A549 and H1435 Xenografts Discussion Figures iii

9 References Appendix iv

10 FIGURE CONTENT Figure 1 Chemical structure indole (A) and 1,1,3-tri(3-indolyl) cyclohexane (3-indole) (B) Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Cytotoxicity of 3-indole in normal human lung fibroblast cells IMR-90 and various human non-small cell lung carcinoma (NSCLC) cells (A549, H1299, H1435, CL1-1, and H1437) In vitro proliferative effects of 3-indole in various human lung cancer cells (A549, H1299, H1435, CL1-1, and H1437 cells) indole induced cell cycle arrest and apoptosis in various human lung cancer cells indole induced G2-M cell cycle arrest in H1437 lung cancer cells Activation of the p53/p21 pathway is required for the induction of cell cycle arrest in 3-indole in various cells indole induced DNA ladder appeared in various human lung cancer cells Effects of 3-indole on the protein levels of Bcl-2, Bax, Bad, caspase-9, -3, -8, and cytochrome c in various lung cancer cells Figure 9 Induction of caspase activity by A549 cells v

11 Figure 10 3-indole induced that loss of the mitochondrial membrane potential (MMP) in A549 cells Figure 11 3-indole induced that mitochondrial aggregates in A549 cells Figure 12 3-indole induced the reactive oxygen species (ROS) production in various lung cancer cells Figure 13 ROS inhibitor reduced the 3-indole-induced ROS accumulation Figure 14 ROS inhibitor reduced the 3-indole-induced apoptosis Figure 15 A time-dependent increase of DNA damage by 3-indole and it can be reversed by adding ROS inhibitor Figure 16 Fold changes of specific genes in A549 cells treated with 3-indole Figure 17 Activation of the JNK signaling pathways is required for the induction of apoptosis in 3-indole treated A549 cells Figure 18 JNK inhibitor (SP600125) reduced the 3-indole-induced apoptosis Figure 19 Effects of 3-indole on the protein levels of Akt (A, B) and COX-2 (C) in various lung cancer cells Figure 20 3-indole effectively inhibited the growth of various human lung cancer cells (A549 and H1435) xenografts vi

12 Figure 21 H&E staining of A549 xenografts Figure 22 Serum biochemistry assays of A549 xenografts Appendix Figure 1 In vitro proliferative effects of 3-indole in various esphogeal cancer cells (KYSE170, KYSE50, KYSE510, and KYSE70) vii

13 ABBREVIATIONS Apaf-1 apoptotic protease activating factor 1 ATF2 activating transcription factor 2 Bcl-2 B-cell leukemia/lymphoma 2 Caspase CAT cysteine-dependent aspartate-specific protease catalase Cdc2 cell division cycle 2 CDK CIP CKI COX DCFH-DA cyclin-dependent kinase cyclin-dependent kinase-interacting protein cyclin-dependent kinase inhibitor cyclooxygenase 2,7 -dichlorofluorescin-diacetate acid DiOC6 DMSO DSB ERK GAPDH GPx HBSS 3,3 -Dihexyloxacarbocyanine iodide dimethyl sulfoxide double-strand block extracellular signal-regulated kinases glyceraldehyde 3-phosphate dehydrogenase glutathione peroxidase Hanks balanced salt solution viii

14 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic INK4 inhibitors of cyclin dependent kinase 4 JNK KIP MAPK MMP MTT c-jun N-terminal kinase cyclin-dependent kinase inhibitory protein mitogen-activated protein kinase mitochondrial membrane potential 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide NAC NF-κB NSCLC PAGE PBS RB ROS SCLC SDS SOD TBE TNF N-acetyl cysteine Nuclear factor-kappa B non-small cell lung cancer Polyacrylamide gel electrophoresis Phosphate-Buffered Salines retinoblastoma Reactive oxygen species small cell lung cancer Sodium dodecyl sulfate superoxide dismutase Tris-Borate-EDTA tumor necrosis factor ix

15 中文摘要 目的 : 肺癌在世界各地無論男性或女性都是發病率 死亡率名列前茅的惡性腫瘤 因此, 發現與合成新穎的肺癌治療抗癌藥物是刻不容緩的工作 材料與方法 : 本研究發展了一種新穎的吲哚結構合成化合物 1,1,3-tri(3-indolyl)cyclohexane (3-indole), 設計使用二步法合成, 該技術方法縮短製備過程, 產品質量和產量也獲得提高, 並藉由人類肺癌細胞株 (A549, H1299, H1435, CL1-1, and H1437) 來探討新穎抗癌藥物對於肺癌細胞的毒殺作用及其機制, 同時進行前臨床動物實驗測試 結果 : 新穎的抗癌藥物 3-indole 經由不同濃度處理, 可以誘導人類肺癌細胞株 (A549, H1299, H1435, CL1-1, and H1437) 進行細胞週期休止 (cell cycle arrest) 及細胞凋亡 (apoptosis) 細胞週期研究初步實驗結果顯示調控細胞週期休止的蛋白 p53 與 p21 表現增加, 顯示 p53/p21 相關訊息傳遞路徑重要性 目前已知有兩個機轉可以調控細胞凋亡現象, 第一個作用機轉是經由 caspases (cysteine-dependent aspartate-specific proteases) 相關性機轉活化而引起細胞凋亡, 目前已被認定有粒線體參與訊息傳遞的內在路徑與細胞外死亡訊息接受器作用的外在路徑 ; 第二個機轉是經由 caspases 非相關性機轉 西方墨點法實驗結果顯示, 調控細胞凋亡進行的促進凋亡蛋白 Bax Bad 表現增加, 抗凋亡蛋白 Bcl-2 表現下降, 而粒線體細胞色素 C 釋放至細胞質情形也有增加, 另外一方面, 透過 caspases 活性分析實驗結果顯示,3-indole 主要是藉由 caspases-9 caspases-3 參與粒線體訊息傳遞的內在路徑以誘發細胞凋亡發生 此外,3-indole 誘導 A549 人類肺癌細胞株粒線體膜電位下降 活性氧分子 (reactive oxygen species, ROSs) 產量增加, 與細胞生長調節相關 MAPK (Mitogen-activated protein kinase) 家族分子

16 c-jun N 端蛋白質激酶 (JNK) 表現增加, 同時顯示有 DNA 損傷情形 進一步活性氧分子抑制劑實驗結果顯示,JNK 表現與 DNA 損傷可部分減少 3-indole 誘導細胞凋亡情形受到活性氧分子抑制劑或 JNK 訊息抑制劑阻斷, 顯示活性氧分子與 JNK 壓力相關訊息傳遞路徑重要性 此外, 初步實驗結果, 其他生長調節相關訊息傳遞蛋白 ( 如 Akt 與 p38/cox-2) 表現也受到 3-indole 抑制, 顯示 PI3K/Akt 與 p38/cox-2 訊息傳遞路徑重要性 同時前臨床動物實驗測試結果顯示 3-indole 抑制 A549 及 H1435 肺癌細胞株生長 結論 :3-indole 在細胞模式與動物模式呈現具有抑制肺癌細胞株生長的作用, 其誘導細胞死亡是透過 ROS 與 JNK 路徑之粒線體訊息傳遞的內在細胞凋亡, 同時可誘導細胞週期休止以及抑制肺癌細胞株 Akt 與 p38/cox-2 的表現, 顯示使用二步法合成, 具有高質量和產量的 3-indole 具有發展作為新穎的抗癌症用藥的價值 2

17 ABSTRACT BACKGROUND. Lung cancer is the most common malignancies in both men and women worldwide. Thus, the development of more effective anti-cancer drugs for lung cancer is urgently needed METHODS. This study generated a 2-step novel synthetic compound, referred to as 1,1,3-tri(3-indolyl)cyclohexane (3-indole), in high purity and yield. 3-indole was tested for its biological activity in A549, H1299, H1435, CL1-1, and H1437 lung cancer cells. Animal studies were also performed. RESULTS: The data indicated that 3-indole induced cell cycle arrest and apoptosis in various lung cancer cells. Increased expression of p53 and p21 protein suggested the importance of p53/p21 pathway in 3-indole-induced cell cycle arrest. Increased cytochrome c release from mitochondria to cytosol, decreased expression of anti-apoptotic Bcl-2, and increased expression of pro-apoptotic Bax and Bad were observed. In addition, 3-indole stimulated caspases-3, -9 and to a lesser extent caspase-8 activities in cancer cells, suggesting that the intrinsic mitochondria pathway was the potential mechanism involved in 3-indole-induced apoptosis. 3-indole-induced a concentration-dependent mitochondrial membrane potential dissipation, and increase in reactive oxygen species (ROSs) production. Activating c-jun N-terminal kinase (JNK) and triggering DNA damage were also apparent. Note that 3-indole-induced JNK activation and DNA damage can be partially suppressed by ROS inhibitor. Apoptosis induced by 3-indole could be abrogated by ROS or JNK inhibitors, suggesting the importance of ROS and JNK stress-related pathways in 3-indole-induced apoptosis. Preliminary data of decrease expression of Akt, p38, and COX-2 proteins 3

18 suggested the importance of PI3K/Akt and p38/cox-2 pathways in 3-indole induced cell apoptosis. Moreover, 3-indole showed in vivo anti-tumor activities against human xenografts in murine models. CONCLUSIONS. The result from the present study suggest that 3-indole inhibited the growth of various human lung cancer cells in cell and animal models and induced intrinsic apoptosis by ROS production and activation of the JNK signaling pathways. Together, these data confirmed that the 2-step synthetic 3-indole compound of high purity and yield is a potential candidate to be tested as a lead pharmaceutical compound for cancer treatment. 4

19 INTRODUCTION I. Outline of lung cancer Lung cancer is the most frequent cause of cancer mortality in the world, in both men and women (Danesi et al., 2003; Jemal et al., 2007). It is extremely difficult to detect lung cancer early for curative treatment. Lung cancer is a lethal disease because of the 5-year overall survival after the initial diagnosis in many countries generally less than 15 % (Danesi et al., 2003). From a histological point of view, lung cancer is classified into small cell lung cancer (SCLC) and non-small cell (NSCLC); 20 % are SCLCs, and 80 % are NSCLCs, including adenocarcinomas, large cell carcinomas, and squamous cell carcinomas in Taiwan (Department of Health, 2007). Lung cancer is notoriously difficult to treat effectively. There are three standard ways to treat lung cancer: surgery, radiotherapy, and chemotherapy. Chemotherapy and radiotherapy have a role to cure locally advanced and metastatic tumors, either as a portion of a treatment strategy or as a mitigate therapeutic approach of choice (Danesi et al., 2003; Pfister et al., 2004; Molina et al., 2006). Even with multi-modality therapies and the recent advent of novel molecular targeted therapies (e.g., epidermal growth factor receptor inhibitors), they can be given both though a vein or as pills by mouth, the clinical responses to chemotherapy in patients with lung cancer are still unsatisfactory (Danesi et al., 2003). Thus, the 5

20 development of novel and more effective anti-cancer drugs for lung cancer is urgently needed. II. Overview of cell cycle 1. Cell cycle The cell cycle is a highly ordered process that results in the duplication and divides into two daughter cells. Cancer cells acquire unlimited replication potential and continue to divide without progressing into immobility and senescence (Hayflick 1997; Sherr 2000; Tyson & Novak 2001). And so to understand cancer we require to know what is cell proliferation and how is it regulated? The eukaryotic cell cycle is divided into two phases, interphase and mitosis. Interphase includes:g1 (gap phase 1), S (DNA synthesis), and G2 (gap phase 2). During G1 phase, the cell is subject to stimulation by extracellular mitogens and growth factors and integrates growth preparation of the chromosomes for replication. S phase is defined as the synthesis of DNA and duplication of the centrosome. G2 phase is a process after S phase that the cell prepares for division. In mitosis (M) phase, the duplicated chromosomes segregate and cell division (Sherr 2000). Finally, there is a fifth state, G0 (also known as quiescence/temporarily or permanently out of cycle) into which the cell may reversibly exit from G1, if it is response to growth or mitotic signals (Lundberg & Weinberg 1999; Israels & Israels 2000; Park & Koff 2001; Murray 2004). 6

21 2. Cell cycle checkpoint Cell cycle events are regulated by a network of many molecular signals at a number of positions within the cell cycle known as checkpoints. Checkpoint as a mechanism for monitoring the integrity of DNA are strategically placed at the G1-S and at the G2-M phase, the events in each phase are complete before moving to the next, that cells with DNA damage do not replicate (Israels & Israels 2000; Park & Koff 2001). Progression through each phase of the cell cycle is regulatory via many molecules, including cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). In general, cell cycle transitions are controlled by CDK family of serine/threonine kinases. These holoenzymes contain both regulatory (cyclin) and catalytic (CDK) subunits that the activity of each of these kinases is dependent on its association with cyclin. Different cyclin/cdk complexes are expressed only in the appropriate phase of the cell cycle, phosphorylate specific protein substrates to move the cell through the cycle, and controlled via degradation by ubiquitin-mediated proteolysis (Sherr & Roberts 1999). The cycle begins in G1, a critical time where extracellular signals both positive and negative, by D cyclins (D1, D2, and D3) associate with CDK4 and CDK6 (Sherr & Roberts 1999). Formation of the cyclin/cdk complexes results in phosphorylation and activation of the CDKs. The activated CDKs can phosphorylate the retinoblastoma (RB) protein causing the E2F transcription factor dissociation of from RB. The activated of E2F can transcribe a number of responder genes (including cyclin E and cyclin A) and promote cell cycle for the 7

22 transition from G1 into S. An important response of the normal cell to DNA double-strand blocks (DSBs) is activation of pathways which induce arrest at the G1-S transition. This is so that cells which are in G1 and have suffered DNA damage do not enter S phase, prevents replication of damaged DNA. DNA damage checkpoints operate in the G1, S, and G2 phases of the cell cycle until the damage is repaired. DNA damage during S phase does not actually stop replication, but instead slows replication if damage has occurred (Rhind & Russell 2000; Heffernan et al., 2002). The cyclin E/CDK2 complex is required for the transition from late G1 into S. Cyclin A is expressed soon after cyclin E at the G1-S boundary. The binding of cyclin A to CDK2 occurs at the G1-S transition and persists through S phase results in DNA synthesis proceeds. The Cdc2 (also known as CDK1) completed with cyclins A and B is required for progression from G2 into mitosis. The cyclin/cdk1 complexes phosphorylate cytoskeleton proteins including lamins, histone H1, and components of mitotic spindle. 3. Cyclin-dependent kinases inhibitors (CKIs) Two families of CDK inhibitors are involved in cell cycle regulation. The first is CIP/KIP (cyclin-dependent kinase-interacting protein/ cyclin-dependent kinase inhibitory protein) family includes the inhibitors p21, p27, and p57. The second family is INK4 (inhibitors of cyclin dependent kinase 4), all contain ankyrin repeats structure, constitutively expressed INK4 genes, includes the inhibitors p16, p15, p18, and p19. The INK4 family of proteins specifically interacts with 8

23 CDK4 and CDK6 but not other CDKs. Cells respond to DNA damage by activating cell cycle checkpoints. Several findings have demonstrated that the product of the p53 tumor suppressor gene is responsible for the G1 checkpoint. Recent observations suggest that p53 also plays a role in regulating the G2-M transition. However, it has also been documented that the G2-M transition may be regulated independently from p53, since cells that are p53 nullizygous or with mutated p53 show a DNA damage-induced G2 arrest (Liebermann et al., 1995; Pellegata et al., 1996). DNA damage induced G1, S, or G2 arrest in many cell types by directly and indirectly activating p53 through inhibition of RB phosphorylation. The CIP/KIP family protein (such as p21) at several sites in the cell cycle, targeting CDKs (4, 6, and 2), is response to upregulation by either p53-dependent or -independent mechanisms. As a result of the action of p21, binds to cyclin D/CDK4/6 is inhibited and arrest of cellular proliferation at the checkpoint in G1 or binds to cyclin E/CDK2 causing arrest at the G1-S transition. The increase in p21 followed by inhibition of CDK4 and CDK6 prevents phosphorylation of RB and, as a result, the cell remains in G1 allowing time for DNA repair (Huang et al., 2001; Sheahan et al., 2007). It has also been reported for nontransformed fibroblast cells that p21 transiently colocalizes with cyclin A or cyclin B1 in the nucleus at G2-M (Dash & El-Deiry 2005). III. Overview of apoptosis 1. Apoptosis Apoptosis, a gene programmed mode of cell death, is a major 9

24 control mechanism by which cells die if DNA damage is not repaired or as a defense mechanism such as in immune reactions, depends on the subsequent activation of different processes involving cellular districts alteration (i.e. cell membrane death receptor activation or mitochondria disorder), inducible protein phosphorylation (i.e. mitogen activated protein kinases or transcription factors), and activation of proteases (Wyllie & Golstein 2001; Broker et al., 2005; Elmore 2007). Apoptosis is also essential that plays an important role in controlling cell populations in tissues and maintenance of cellular homeostasis mechanism as regulator of normal development and aging. 2. Pathways of Apoptosis Apoptosis occurs through two main pathways. The first, referred to as the extrinsic pathway involve transmembrane receptor-mediated interactions, is triggered through the Fas death receptor, a member of the tumor necrosis factor (TNF) receptor superfamily (Zapata et al., 2001; Elmore 2007). The second pathway is the intrinsic or mitochondrial pathway that initiate apoptosis involve a diverse array of non-receptor-mediated stimuli that produce intracellular signals. The stimuli that initiate the intracellular signals that disruption of the mitochondrial potential across the inner membrane and the release of cytochrome c from the mitochondria to the cytosol (Liu et al., 1996; Susin et al., 1999; Desagher & Martinou 2000; Du et al., 2000). During apoptosis have identified the various changes: (1) shrinkage, the cells are smaller in size; (2) pyknosis, the result of chromatin condensation; 10

25 (3) DNA fragmentation, the endonuclease cleavage at the linker regions of histone-dna complex, products of apoptosis; and (4) extensive plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies that consist of cytoplasm with tightly packed organelles with or without a nuclear fragment. In addition, the cellular distribution of mitochondria is profoundly affected during apoptosis. Mitochondria are normally dispersed throughout the entire cell. One of the early events that occurs during apoptosis, Both mitochondrial condensation and perinuclear clustering can be observed in many cell types (Desagher & Martinou 2000). Apoptotic cells do not release their cellular constituents into the surrounding tissue and apoptotic bodies are recognized and removed by phagocytic cells and thus apoptosis is also notable for the absence of inflammation around the dying cell. 3. Caspases (cysteine-dependent aspartate-specific proteases) Apoptosis alteration pathways converge to a final common pathway involving the activation of a cascade of proteases called caspases (cysteine-dependent aspartate-specific proteases). Caspases are a family of specific cysteine proteases being the key effector molecules in apoptosis (Goodsell 2000; Wyllie & Golstein 2001; Broker et al., 2005). They usually exist in cells as pro-caspase (inactive zymogen) and are activated by proteolytic cleavage into constituent subunits. After cleavage the pro-caspase molecule reconstitutes as a heterodimer consisting of a large and small subunit and two heterodimers function as 11

26 the active caspase. Once activated, initiator caspases (upstream caspases -8 and -9) activate effector caspases (downstream caspases -3, -6, and -7), resulting in an amplification of the caspase cascade. Caspases also affect cell cycle regulation, cell signaling pathways, and cell cytoskeletal structure, ultimately leading to the morphologic appearance of apoptosis, such as chromatin condensation and DNA fragmentation. 4. Bcl-2 family The Bcl-2 family proteins are critical mediators of the mitochondrial pathway of apoptosis that control the release of apoptosis molecules from the mitochondria (Danial & Korsmeyer 2004). Some of these proteins (such as Bcl-2 and Bcl-xl) are anti-apoptotic, while others (such as Bax and Bad) are pro-apoptotic. In addition, the regulation of apoptosis is through heterodimerization of anti-apoptotic and pro-apoptotic members of the Bcl-2 family. One of the interesting aspects of apoptosis regulation by members of the Bcl-2 family is their subcellular localization and translocation. Bax is a pro-apoptotic member of the Bcl-2 protein family that is mainly localized in the cytosol of healthy cells and translocates to mitochondria after a variety of death stimuli (Murphy et al., 2000). Bad translocates to the outer membrane of mitochondria during apoptosis induced in cells starved of growth factors (e.g. interleukin-3), where they inactivate the anti-apoptotic Bcl-2 family proteins Bcl-xl and Bcl-2 or activates pro-apoptotic Bax (Porter 1999). Furthermore, Bad phosphorylation by Akt inhibits its pro-apototic effects, which Akt-mediated 12

27 phosphorylation results in substrate inhibition through the regulation of subcellular localization by interaction with proteins (i.e. Bad) (del Peso et al., 1997; Porter 1999). The principal mechanism by which Bcl-2 family proteins regulate apoptosis is probably by controlling cytochrome c release (Desagher & Martinou 2000). Once released from mitochondria, cytochrome c forms a multiprotein complex (apoptosome) with Apaf-1 and procaspase -9, leading to activation of this initial caspase and induction of downstream apoptotic protease cascade (Li et al., 1997). IV. Reactive oxygen species (ROS) Reactive oxygen species (ROS), include the superoxide anion, hydrogen peroxide, and the hydroxyl radical, which are the products of endogenous cellular oxidative processes and exposure to exogenous agents (such as H 2 O 2 ), and increased by the imbalance of the redox status or inhibited by antioxidants (Chrestensen et al., 2000; Koren et al., 2001). Two systems of antioxidants are involved in the imbalance of the redox status. The first is enzymatic antioxidant such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). The second family is nonenzymatic antioxidant inluding tocopherols (vitamin E), ascorbic acid (vitamin C), and carotene. One of the major sources of endogenous ROS is mitochondrial electron transport chain (Raha & Robinson 2000). The ROSs have been shown to induce various biological processes [(such as activation of protein tyrosine kinase and the stimulation of downstream proteins including mitogen-activated 13

28 protein kinases (MAPK)], including post-transcriptional or post-translational modifications of many genes, which lead to diverse outcomes, such as cell growth, cell differentiation, and apoptosis (Dougherty et al., 2004; Zhou et al., 2006). The mitochondrial membrane has been shown to be sensitive to the redox state and ROS may play an importance regulator in apoptosis, either as activators of mitochondrial membrane permeabilization or a consequence of this transition, depending on the death stimulus. Oxidative damage results in a significant premonition to cellular integrity in terms of damage to DNA (such as single- and double-strand breaks), proteins, lipids, and other macromolecules. However, there is now forceful evidence that ROSs are not only toxic effect of cellular metabolism but also essential molecules in cellular signaling and regulation (Reth 2002; Chiarugi & Cirri 2003). In this context, it has been reported that apoptosis induced by chemotherapy compounds is dependent on ROS production and its interaction with other signal transduction cascades molecules in apoptosis and cell cycle arrest (Filomeni et al., 2003). V. The mitogen-activated protein kinase (MAPK) family The mitogen-activated protein kinase (MAPK) signaling family, a family of serine/threonine kinases, transduces signals that regulate proliferation, differentiation, survival and apoptosis (Roux & Blenis 2004). The best characterized members of the MAPKs include the extracellular signal-regulated kinases (ERK1/2), the p38 kinases (p38α/β/γ/δ), and the c-jun N-terminal kinase/stress-activated protein 14

29 kinases (JNK1/2) that can be activated by growth factors, DNA damage, cytokines, oxidant stresses, UV light, anticancer drugs, and osmotic shock (Johnson & Lapadat 2002; Olson & Hallahan 2004). ERK is generally considered to be a survival mediator involved in the protective actions of growth factors in apoptosis, whereas JNK and p38 are usually referred to as stress-stimulated MAPKs, which are required for the induction of apoptosis by diverse stimuli such as chemical stresses, oxidants, and inflammatory cytokines (Kyriakis & Avruch 2001). Notably, p38 and JNK are themselves phosphorylated and activated by upstream kinases. JNK activation of c-jun is necessary for apoptosis in myeloid and lymphoid cells, because use of a dominant-negative c-jun mutant blocks programmed cell death in these cells (Lei et al., 2002). Even though transcription factors are targets of MAPK, only part of the MAPK translocates to the nucleus and quantity remains in the cytoplasm to regulate post-transcriptional modification involving cytoplasmic targets gene expression. For example, JNK has been reported to catalyze the phosphorylation of Bcl-2 and Bad promotes the apoptotic effect that directly links the JNK signaling pathway to the cell death machinery (Fan et al., 2000; Donovan et al., 2002). The p38 in turn stimulates the activity of several transcription factors including ATF2 and Elk-1. Recent studies have shown that MAPKs signaling pathways regulate the cell cycle. p38 kinase has been demonstrated as essential for sustained G2 arrest induced by γ-irradiation, vanadate, and genistein (Wang et al., 2000; Frey & 15

30 Singletary 2003; Lavelle et al., 2003; Zhang et al., 2003). Activation of different p38 MAPK isoforms has been suggested that p38α is proapoptotic, whereas p38β is anti-apoptotic in neonatal rat cardiomyocytes (Sugden & Clerk 1998; Wang et al., 1998b). However, there have also been reports indicating that the p38 is associated with the development of chemoresistance by activating NF-κB (Hendrickx et al., 2003; Yu et al., 2004). Furthermore, Several proinflammatory treatments which induce COX-2 gene expression also stimulate p38. VI. Compounds with an indole structure 1. Microtubule structure and function Natural and synthetic compounds with an indole structure have been shown to induce apoptosis through cell cycle arrest or a cellular stress activation mechanism. A number of anti-microtubule compounds characterized by the presence of an indole core nucleus have been obtained (Brancale & Silvestri 2007). Microtubules are main components of the cytoskeleton and are important for a variety of cell functions, including maintenance of cell shape, transportation of vesicles, mitochondria and other components throughout cells, and segregation of chromosomes during cell division (Jordan & Wilson 2004; Pellegrini & Budman 2005). Microtubules are extremely dynamic polymers consisting of α tubulin and β-tubulin heterodimers arranged in the form of slender filamentous that constantly assembly (polymerization) or shortening (depolymerization) (Jordan 2002). Cancer cells acquire unlimited replication potential and continue to 16

31 divide without progressing into immobility and senescence (Hayflick 1997). The properties of uncontrolled proliferation and division make cancer cells extremely dependent upon the high dynamics of microtubule and hence sensitive to anti-microtubule compounds (Jordan & Wilson 1998). Anti-microtubule agents (with various tubulin-binding sites), which have been found to interfere with tubulin/microtubules dynamic equilibrium, induce G2-M cell cycle arrest and trigger apoptosis (Woods et al., 1995; Jordan et al., 1996). These findings indicate that microtubule is an important target for the development of novel anticancer drugs (Giannakakou et al., 2000). 2. Anti-microtubule drugs The clinically used anti-microtubule drugs generally fall into two main groups. One group includes vinca alkaloids, known as the microtubule-destabilizing agents such as vinorelbine, vincristine, and vinblastine. This type of agent inhibits microtubule polymerization and lead to the depolymerization of existing microtubules. The other group is known as the microtubule-stabilizing agents, including taxanes, such as taxol (paclitaxel) and docetaxel, stabilize microtubules and induce a net polymerization (Li & Sham 2002). Furthermore, synthetic indole structure compounds (such as ABT-751 and BPR0L075) can cause G2/M arrest and apoptosis (Wang et al., 1998a; Kuo et al., 2004; Yee et al., 2005). Despite the efficiency of anti-microtubule drugs in inhibiting the progression of some tumors, the important unsolved questions about the anti-tumor activities of anti-microtubule drugs concern the basis of 17

32 their tissue specificities in many cancer types and the basis for the development of drug resistance to these agents usually occur during therapy (Gottesman 2002). VII. 1,1,3-tri(3-indolyl)cyclohexane (3-indole) Indole-3-carbinol (I3C) and 3,3 -diindolylmethane, which are phytochemicals commonly found in cruciferous vegetables, induce G1 cell cycle arrest and apoptosis mediated by alterations in stress-activated protein kinase and activation of a DNA damage mechanism (Hong et al., 2002; Brew et al., 2006; Gong et al., 2006). However, these natural and semi-synthetic indole compounds have some disadvantages, such as harsh reaction conditions, long reaction times and expensive preparation. The study recently developed a novel 2-step synthesized indole compound, 1,1,3-tri(3-indolyl)cyclohexane (3-indole), in high purity and good yield (Ko et al., 2006). In the present study, the biological activities especially the mechanisms involved in the anti-cancer growth activities of 3-indole in cell and animal models were evaluated. 3-indole induced G1 cell cycle arrest at low concentration (10 μm), except for H1437, apoptosis at high concentration (30 μm) in most human lung cancer cell lines (A549, H1299, and CL1-1), and the partially G2-M arrest of 30 μm of 3-indole in H1437. We also found that 3-indole induced G1 cell cycle arrest maybe associated with p53-dependent or independent regulation of p21 expression. Furthermore, we found that 18

33 apoptosis was induced via an intrinsic mitochondrial pathway involving stress-activated pathways, including ROS and JNK activities. The events of apoptosis induced by 3-indole, such as mitochondrial membrane potential (MMP) dissipation, Bcl-2 inactivation, cytochrome c release, and DNA ladder were observed. Moreover, in vivo anti-tumor activities against human xenografts in murine pre-clinical models indicated that 3-indole is a potential candidate to be tested as a lead pharmaceutical compound for cancer treatment. 19

34 MATERIALS AND METHODS I. 1,1,3-tri(3-indolyl)cyclohexane (3-indole). The compound 1,1,3-tri(3-indolyl)cyclohexane (3-indole) was synthesized at the Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan, Republic of China. 3-indole was obtained as a solid powder in ~ 90 % yield. 3-indole was dissolved in 100 % dimethyl sulfoxide (DMSO) before further dilution in cell culture medium. The detailed synthetic method was described in our previous paper by Ko et al. (Ko et al., 2006). II. Cell Culture. Human non-small cell lung cancer (NSCLC) cells (A549, H1299, CL1-1, and H1437) were maintained in DMEM and human NSCLC H1435 cells were maintained in RPMI 1640 medium. Human esophageal cancer cells (KYSE170, KYSE50, KYSE510, and KYSE70) were maintained in RPMI 1640 medium. All media were supplemented with 10 % fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Eugene, Oregon, USA). The cells were maintained at 37 o C in a humidified incubator containing 5 % CO 2 in air. III. Cell Proliferation Assay. Cells were seeded at cells/well in 6-well plates and treated with solvent control DMSO or various concentrations of 3-indole for the indicated times. During the last 30 minutes of treatment, the cells were treated with 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). 20

35 Cells having functional succinate dehydrogenase of mitochondria can convert MTT to formazan that generates blue color when dissolved in DMSO. The intensity was measured using a reader for enzyme-linked immunosorbent assay and an absorption wavelength of 540 nm. Cell proliferation results are expressed as percentage loss of cell viability compared with DMSO control. IV. Analysis of Cell Cycle Distribution. The assay was performed according to Kuo et al. (Kuo et al., 2004). Cells were incubated with various concentrations of 3-indole (0, 10, or 30 μm) for 24 or 48 h. Adherent and floating cells were collected, washed once in 1 Phosphate-Buffered Salines (PBS), and fixed with ice-cold 80 % ethanol for at least overnight at 20 C until analysis. Fixed cells were collected by centrifugation, washed once with 1 PBS, resuspended in 1 ml of 1 PBS (contains 20 μg/ml propidium iodide, 200 μg/ml RNase A, and 0.1 % triton X-100) and then incubated in the dark for 10 minute. Determination of cell cycle distribution was performed by FACScan flow cytometer (BD, MountainView, CA) and calculated using ModFIT LT 2.0 version software (BD). V. Determination of the Apoptotic DNA Ladder. Fixed cells (as described in the Cell Cycle section) were collected by centrifugation, resuspended in 100 μl of DNA extraction buffer (0.2 M Na 2 HPO 4, 0.1 M citrate acid, and 0.5 % triton X-100, ph 7.8), and then incubated for 1 h at 37 C. After centrifugation, the supernatant was collected and incubated with 5 μl RNase A (100 mg/ml) for 1 h at 37 C, and followed by digestion with 5 μl proteinase K (20 mg/ml) for 1 h at 37 C. 21

36 Horizontal electrophoresis of the samples and molecular marker was performed with a 2 % agarose gel and 0.5 TBE buffer at 50 V, using a MUPID-2 mini electrophoretic system (Cosmo Bio Co., Tokyo, Japan). After electrophoresis, the gels were stained with ethidium bromide and the fragmented DNA imaged using a FluorImager (Amersham Biosciences, Tokyo, Japan). VI. Evaluation of the Mitochondrial Transmembrane Potential. The assay was basically performed according to the method described by Kuo et al. (Kuo et al., 2004). The cationic fluorescent probe 3,3 -Dihexyloxacarbocyanine iodide (DiOC6) (Invitrogen) was used to monitor drug-induced changes in the mitochondrial transmembrane potential. Cells were initially seeded at cells in 6-well dishes and then treated with various concentrations of 3-indole (0, 2, 10 or 30 μm) for the indicated time. After drug treatment, the cells were treated with the probe DiOC6 (40 nm) in appropriate medium for 30 minutes at 37 C before cytometric analysis. The cells were collected by centrifugation, washed once with 1 PBS, resuspended in 1 PBS. Measurement of the retained DiOC6 in 10,000 cells of each sample was performed in a FACScan flow cytometry (BD). VII. Western Blot Analysis. Cells were initially seeded at a density of in 100-mm 2 dishes. After treated with various concentrations of 3-indole (0, 10, or 30 μm) 3-indole for the indicated time, adherent and floating cells were collected, washed once in 1 PBS, and lysed in ice-cold lysis buffer (0.5 M Tris-Hcl (ph 7.4), 1.5 M NaCl, 2.5 % deoxycholic acid, 10 mm EDTA, 10 % NP-40, 0.5 mm DTT, 1 mm 22

37 phenylmethylsulfonyl fluoride, 5 ug/ml leupeptin, and 10 μg/ml aprotinin). The lysate was centrifuged at rpm for 30 min at 4 C and protein content of the supernatant was measured. Cell lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes. Membranes were blocked and probed with appropriate dilutions of primary antibody, as recommended by the manufacturers. The primary antibodies used were p53, p21, caspase-3, caspase-8, caspase-9, cytochrome c, p44/42 MAPK, phospho-p44/42 MAPK, JNK, phospho-jnk, p38, phospho-p38, phospho-akt, COX-2 (all from Upstate Biotechnology Inc., Lake Placid, NY), Bcl-2, Bad, and Bax (all from Chemicon Inc.), phospho-c-jun (Cell Signaling Technologies, Beverly, MA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Novus Biologicals, Littleton, CO). Membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized using Western blot chemilluminescent reagents. VIII. Determination of Caspase Activity. Caspase activity was measured with the caspase colorimetric assay kit (BioVision, Mountain View, CA) according to manufacturer's instructions. After treatment, cells were lysed and the cell lysates were incubated with various synthetic caspase substrates (Ac-DEVD-pNA, Ac-LETD-pNA, and Ac-LEHD-pNA) to measure the activity of caspases -3, -8, and -9, respectively. The equivalent of cell lysates (150 μg of protein) was pipetted into each well of a 96-well plate and 50 μl of reaction buffer (100 mm HEPES, ph 7.4), 20 % v/v glycerol, 0.5 mm EDTA, 5 mm 23

38 DTT (added freshly) containing caspase substrate (final concentration 4 mm) were added. Following incubated at 37 C and measure the absorbance at 405 nm every 2 h until 72 h. IX. Immunocytochemistry. The localization of mitochondria was detected using MitoTracker (Invitrogen) as a fluorescent probe. Cells were initially seeded at a density of in 60-mm 2 dishes. Cells were treated with 0 or 30 μm 3-indole for the indicated times. During the last 30 minutes of treatment, cells were treated with the MitoTracker (20 nm). Cellular mitochondria were observed with an Olympus BX50 fluorescence microscope (Optical Elements Corporation, Dulles, VA). X. Determination of Intracellular Reactive Oxygen Species. The assay was performed as described by Juang et al. (Juang et al., 2007). The intracellular level of ROS (reactive oxygen species) was measured using DCFH-DA (2,7 -dichlorofluorescin-diacetate) (Invitrogen) as a fluorescent probe. Cells were initially seeded at cells in 6-well dishes and then treated with various concentrations of 3-indole (0, 10 or 30 μm) 3-indole for the indicated time. After drug treatment, the cells were treated with the probe DCFH-DA (10 μm/ml) in appropriate medium for 30 minutes at 37 C before cytometric analysis. The cells were collected by centrifugation, washed once with 1 PBS, resuspended in 1 PBS. Measurement of the retained DCFH-DA in 10,000 cells of each sample was performed in a FACScan flow cytometry (BD). 24

39 XI. Pulsed-Field Gel Electrophoresis. The assay was performed according to the method described by Juang et a. (Juang et al., 2007). Cells were collected and resuspended in 1 PBS. PBS-suspended cells were mixed with 1 % low-melting point agarose solution at a final concentration of cells per 0.1 ml of agarose block. The mixture was immediately poured into molds, then embedded at 4 o C for 30 min. The agarose-embedded cells were digested in lysis buffer containing 25 mm of EDTA, 10 mm of NaCl, 1 % N-Lauroyl sarcosine sodium, 0.1 % SDS, 10 mm of Tris-HCl (ph 8.0), and 1 mg/ml of proteinase K for 24 hr at 55 o C, followed by washing with TE buffer (10 mm Tris-HCl (ph 7.5), 1 mm EDTA, and 1 μg/ml RNase A) for 3 h with buffer changed each hour. The agarose plugs containing purified DNA were inserted into 1 % agarose gels and the DNA was analyzed by pulsed-field gel electrophoresis using a FIGE Mapper Electrophoresis System (Bio-Rad) for 16 h at 12 o C. After electrophoresis, the gels were stained and imaged. XII. cdna Microarray Analysis. Untreated A549 cells and A549 cells treated with 30 μm 3-indole for 4, 8, and 12 h were analyzed for 3-indole induced mrna expression profile by cdna microarray. Experimental A549 RNA was isolated using Trizol reagent (GIBCO BRL, Life Technology, USA). From each sample, total RNA (Control universal human reference RNA (Stratagene, La Jolla, CA) and experimental A549 RNA were used to generate cdna. Microarray slides were scanned using GenePix 4000B Biochip Analyzer (Axon Instruments, Union City, USA). Changes in gene expression were 25

40 presented as logarithmic ratios of fluorescence intensities. The logarithmic ratios of each indicated times were then normalized for each gene to that of Control RNA to obtain the expression pattern (the log-intensity log2r of the red dye versus the log-intensity log2g of the green dye, as well as the log intensity ratio M= log 2 R/G, experimental Cy5 and Control Cy3). The genes that showed substantial differences after drug treatment were selected based on at least a 2-fold change in expression. XIII. Subcutaneous Implantation of Cancer Cells in Animals and Monitoring of in Vivo Anti-tumoral Activity after Drug Treatments. Athymic nu/nu female mice (ICR-Foxn1), 4 5 weeks of age, were obtained from the National Laboratory Animal Center (Taiwan, Republic of China). The animals were implanted subcutaneously (s.c.) with A549 or H1435 lung cancer cells in 0.1 ml Hanks balanced salt solution (HBSS) in one flank per mouse. The size of the tumor mass was measured and the tumor volume was calculated as 1/2 length width 2 in mm 3. In human lung cancer xenograft studies, when tumors attained a mass of ~ 50 mm 3, animals were treated intraperitoneally (i.p.) with 3-indole at 0.2 mg/day on days 0, 2, 4, 6, and 8 (final dose, 50 mg/kg) or 0.1 mg/day on days 0, 2, 4, 6, and 8 (final dose, 25 mg/kg), or a solvent (control). A solvent mixture contains DMSO/Cremophor EL/saline (2:1:7). The assay solvent was basically performed according to the method described by Kuo et al. (Kuo et al., 2004). Tumor size was measured after drug treatment. Prior to being sacrificed, the animals were anesthetized and blood samples 26

41 were collected by intracardiac puncture for the mice organ function test. Before organ dissection, the animals were sacrificed by cervical dislocation. Tumor samples and mice organ tissues (including the lungs and kidneys) were resected, fixed with formalin and embedded in paraffin for histologic examination, stained with hematoxylin and eosin for microscopic evaluation, and examined by a pathologist. 27

42 RESULTS I. 3-indole Apparently Inhibited Growth at Low Concentration and Promoted Cell Death at High Concentration in Various Human Lung Cancer Cells. 1,1,3-tri(3-indolyl)cyclohexane (3-indole) is a novel, 2-step synthetic indole compound of high purity and yield. Its structure is shown in Fig. 1. To test the cytotoxicity effect and future clinical usage of 3-indole, normal human lung fibroblast cells IMR-90 and various human non-small cell lung carcinoma (NSCLC) cells with different p53 status including A549 (p53-wild), H1299 (p53-null), H1435 (p53-mutation), CL1-1 (p53-mutation), and H1437 (p53-mutation) cells were tested. Cells were treated with 0, 1, 2, 5, or 10 μm of 3-indole for 24 h and assessed cell viability by the MTT assay. Fig. 2 shows that 3-indole caused a concentration-dependent reduction in cell viability. 3-indole could achieve an inhibitory concentration (IC) 50 value at ~ 10 μm in various human NSCLC cells (A549, H1299, H1435, CL1-1, and H1437 cells), whereas did not show apparent cycotoxicity to the IMR-90 cells at this concentration. 3-indole efficacy was similar against all human NSCLC cells tested regardless of the status of p53, which is the most common genetic alteration in human cancers. These results show that 3-indole were efficacious against human NSCLC cells with various status of p53. The anti-cancer efficacy of 3-indole was also noted in various human esophageal squamous cell carcinoma cell lines, including KYSE170, KYSE50, KYSE510, and KYSE70 (appendix Figure 1). Furthermore, to examine whether the cytotoxicity observed for 3-indole was due to cell growth inhibition or 28

43 cell death, various human lung cancer cells including A549, H1299, H1435, CL1-1, and H1437 were treated with 0, 2, 10, or 30 μm of 3-indole for the indicated times and cell proliferation was assessed by the MTT assay. Fig. 3 shows that 3-indole caused a concentration-dependent reduction in cell proliferation with apparent inhibition of growth at low concentration (10 μm) and promotion of cell death at high concentration (30 μm) in various human lung cancer cells. II. 3-indole Induced Cell Cycle Arrest and Apoptosis in Various Human NSCLC Cells. Microtubules are highly dynamic polymers composed of α tubulin and β-tubulin heterodimers that constantly assembly (polymerization) or shortening (depolymerization). Indole-like compounds are known to arrest cells in G1 or G2-M, and substantially induce apoptosis (Brandi et al., 2003; Kuo et al., 2004). To determine whether the anti-cancer effect of 3-indole was associated with cell cycle deregulation, the cell cycle distribution was analyzed by flow cytometry, we investigated whether cell cycle arrest and/or apoptosis could be induced in various human lung cancer cells (A549, H1299, H1435, CL1-1, and H1437 cells) treated with 3-indole at 0, 10, and 30 μm for 24 or 48 h. Flow cytometry indicated that 10 μm of 3-indole caused most cancer cell lines, except for H1437, to accumulate in G1 phase and a substantial increase in the sub-g1 region (an apoptosis indicator) resulted from treatment with 30 μm of 3-indole at 24 h (Fig. 4). Furthermore, the partially G2-M arrest efficacy of treatment 3-indole with 30 μm at 48 h was also noted in H1437 cells (Fig. 5). 29

44 III. Activation of the p53/p21 Pathway Is Required for the Induction of G1 Cell Cycle Arrest in 3-Indole. The function of p53 as a tumor suppressor has been demonstrated by experiments showing that p53 correlates with G1 or G2 cell cycle regulation after DNA damage (Kastan et al., 1991; Liebermann et al., 1995; Park et al., 2001; Liu et al., 2003). Some report has been demonstrated that p21, a tumor suppressor, is response to upregulation by p53 or by p53-independent remains cell cycle in G1 or G2-M allowing time for DNA repair. Therefore, we performed Western blot to confirm whether the p53/p21 pathway was activated after 3-indole treatment. The preliminary data in Fig. 6A shows that treatment A549 cells (p53-wild) with 10 μm 3-indole increased the expression of p53 protein after 2 h and subsequently p21 increase expression in 8 h. The similar observations were also noted in H1437 cells (p53-mutation) (Fig. 6B). Furthermore, treatment with 10 μm 3-indole can induced cyclin B1 (an G2-M indicator) expression partial increases at 48 h in H1435 cells (p53-mutation) (Fig. 6C). Interestingly, 3-indole treatment for 12 h initially decreased cyclin B1 (Fig. 6C). IV. 3-indole Induced Apoptosis Through the Activation of the Intrinsic Mitochondrial Pathway. To confirm that the sub-g1 region was caused by apoptosis, we performed a DNA ladder analysis, and found that ladders appeared in various human NSCLC cells (A549, H1299, H1435, and CL1-1 cells) at 24 h, and in H1437 cells at 48 h after 3-indole treatment (Fig. 7). These results suggest that 3-indole maybe induce cell death via G1 or G2-M arrests in various human 30

45 NSCLC cells. Furthermore, using Western blot analysis to investigate the mechanism of 3-indole induced apoptosis, we found that treatment of A549 cells with 30 μm of 3-indole resulted in a time-dependent reduction in the levels of the anti-apoptotic protein, Bcl-2. At the same time, the level of the pro-apoptotic protein, Bax and Bad, was concomitantly increased compared with the cells that were not treated with 3-indole (Fig. 8A upper panel). The expression decrease of Bcl-2 was also noted in H1437 cells (Fig. 8B lower panel). To further dissect the apoptosis pathway induced by 3-indole, we performed Western blot analysis for cytochrome c release and caspase protein expression, and used different fluorogenic tetrapeptide substrates (Ac-DEVD-pNA, Ac-LETD-pNA, and Ac-LEHD-pNA) to measure the activity of caspases -3, -8, and -9, respectively. 3-indole increased the release of cytochrome c from mitochondria to cytosol in 8 h and stimulated caspases -3, -9 (an indicator of the intrinsic mitochondria pathway) and to a lesser extent caspase -8 (an indicator of the extrinsic membrane receptor pathway) activities in A549 cells (Figs. 8 and 9). Together, these results showed that 3-indole induced the execution of apoptosis through the activation of the mitochondrial pathway. V. 3-indole Induced Cell Cycle Arrest and Apoptosis by Reactive Oxygen Species Production and DNA Double-Strand Breaks in A549 or H1299 Cells. A number of studies have shown that loss of the mitochondrial membrane potential (MMP) in cells triggers mitochondrial disruption and the generation of reactive oxygen species (ROSs) (Herrera et al., 2001; Gupta et al., 2003; Gong et al., 2006). ROSs are known to 31

46 damage many molecules including proteins, RNA, and DNA (Salmon et al., 2004; Pan et al., 2005). We examined the changes in the MMP and mitochondrial localization using DiOC6, a cationic fluorescent probe. A concentration-dependent change in MMP was observed at min in A549 cells (Fig. 10, upper panel). The data in Fig. 10 lower panel shows that Treatment of 10 or 30 μm of 3-indole decreased the MMP in 4 h but only high concentration (30 μm) of 3-indole continuously decreased the MMP. Moreover, using cell fluorescence staining, mitochondrial localization was detected by a MitoTracker. In untreated cells, mitochondria were evenly distributed in the cytoplasm. In 3-indole (30 μm) treated A549 cells, aggregated mitochondria increased after 12 h, and dendrite-like structures disappeared (Fig. 11). Next, we examined the changes in ROS production and DNA damage in cells treated with various concentration of 3-indole for the indicated times. A significant increase in ROS production was observed in various human NSCLC cells A549 and H1299 at 2 h with 10 μm 3-indole (Fig. 12). In addition, A549 cells were treated with 3-indole and rotenone (0.05 μm, an inhibitor of mitochondrial respiratory chain complex I) or 3-indole and N-acetylcysteine (NAC) (5 mm, a hydroxyl radical scavenger). The results indicated a partial reversal of ROS production by rotenone (Fig. 13) and reduced apoptosis during co-treatment with rotenone or NAC compared to 3-indole treatment alone (Fig. 14). Since there was an increase in ROS production, we decided to assess the degree of DNA strand break damage using pulsed-field gel electrophoresis (PFGE). A549 cells, following 30 μm 3-indole treatment, exhibited a change in DNA damage at 24 h (Fig. 15 left panel). In 32

47 addition, we treated A549 cells with both 3-indole and rotenone (0.05 μm). The data indicated that co-treatment with rotenone reduced DNA damage compared to 3-indole treatment alone (Fig. 15 right panel). VI. cdna Microarray Analysis to Search For Differential Expressed Genes After 3-indole Treatment. To reveal more potential targets and pathways involved in 3-indole treatment, we performed cdna microarray analysis on untreated A549 cells at 0 h and A549 cells treated with 30 μm of 3-indole at 4, 8, and 12 h and harvested the RNA. The dose chosen were close to the dose needed for apoptosis induction. The rationale for the indicated times was to capture the expression profiles of genes that involved in the apoptotic processes. We found many differentially expressed genes, which are related to cell cycle, apoptosis, and cell signaling pathways (Fig. 16). For example, we found that 30 μm of 3-indole caused changes in the mrna levels of several mitogen-activated protein kinase (MAPK) signaling proteins such as p38β and JNK2. Further, that 3-indole affected other signaling pathways, such as PI3K-Akt and Wnt signaling pathways. In addition, 3-indole treatment reduced the expression of histone deacetylase 1 (HDAC1). VII. Activation of the JNK Signaling Pathways Is Required for the Induction of Apoptosis in 3-indole Treated A549 Cells. cdna microarray data revealed that expression of several proteins in MAPK pathway changed after 3-indole treatment. In addition, ROS has been shown to induce various biological processes, including activation of the 33

48 MAPK pathway (Kamata et al., 2005; Gong et al., 2006). Therefore, we performed Western blot to confirm whether the MAPK signaling pathway was activated after 3-indole treatment and whether ROS was involved in 3-indole induced MAPK activation. Cell lysates were subjected to Western blot analysis using anti-phospho-mapk antibodies (ERK1/2, JNK, and p38) to detect phosphorylated activated MAPK family proteins. The data in upper panel of Fig. 17 shows that 3-indole increased the protein level of phosphor-jnk1 in 4 h and phosphor-jnk2 in 8 h. In addition, we found that 3-indole increased the phosphorylation of c-jun, a major nuclear factor of the JNK signaling pathway in 4-12 h. Furthermore, we co-treated A549 cells with rotenone (0.05 μm), SP (20 μm, an inhibitor of JNK) or U0126 (10 μm, an inhibitor of ERK). The results indicated that co-treatment of 3-indole with rotenone or SP reduced protein level of phosphor-jnk and c-jun protein expression compared to 3-indole treatment alone (Fig. 17 lower panel). The data in Fig. 18 shows that treatment of A549 cells with a combination of the JNK inhibitor and 3-indole, caused a significant reduction in 3-indole-induced apoptosis when compared to the cells treated with 3-indole alone, whereas no effect of ERK inhibitor on 3-indole-induced apoptosis was seen. In addition, co-treatment with 3-indole and SP reduced DNA damage compared to 3-indole treatment alone (Fig. 15 right panel). The results indicated that inhibition of JNK activation protects against the cytotoxic effects of 3-indole and that ROS may play a role in JNK activation. In addition, we found that 3-indole decreased other growth singling pathway relation protein such as COX-2 and Akt (Fig. 19). The preliminary data indicated that that 3-indole is a multi-target inhibitor 34

49 compound for cancer treatment. VIII. 3-indole Effectively Inhibited the Growth of Human A549 and H1435 Xenografts. To examine whether 3-indole treatment inhibited A549 cell growth in vivo, we followed the tumor growth in 3-indole and vehicle-treated animals (ICR-Foxn1). 3-indole was dissolved in Solvent (a vehicle mixture of DMSO/Cremophor EL/saline, 2:1:7) before further treatment. To further determine the effect of 3-indole over an extended treatment period, tumor size was measured in each animal. In the meantime, 3-indole-treated animals were sacrificed and processed for evaluation of any possible changes in histopathology and serum biochemistry. Fig. 20 shows the tumor growth in solvent-treated animals (control) compared with 3-indole treatments. Treatment with 3-indole (final dose of 25 or 50 mg/kg i.p.) resulted in tumor growth inhibition, compared to that produced by solvent (control) treated animals bearing A549 cell xenografts (Fig. 20 upper panel). The same observations were also noted in a H1435 cell xenograft model (Fig. 20 lower panel). Evaluation of numerous histologic sections of these tissues from animals bearing human A549 xenografts did not indicate any detectable pathologic abnormalities, as examined by H&E staining (Fig. 21). In addition, 3-indole therapy caused no detectable toxicity on tissues and did not affect organ functions. The organ functions tests included liver function tests, such as glutamic oxalacetic transaminase (GOT), glutamic pyvuvic transaminase (GPT), and albumin levels, and renal function tests, such as blood urea nitrogen (BUN) 35

50 and creatinine levels. The organ functions were similar between the 3-indole-treated and the vehicle-treated groups (Fig. 22). 36

51 DISCUSSION We evaluated the biological activities, especially the mechanisms, involved in the anti-cancer growth of 1,1,3-tri(3-indolyl)cyclohexane (3-indole) in cell and animal models. 3-indole caused a concentration-dependent reduction in cell viability. 3-indole could achieve an IC50 value at ~ 10 μm in various human NSCLC cells (A549, H1299, H1435, CL1-1, and H1437 cells), whereas did not show apparent cycotoxicity to the IMR-90 cells. The anticancer efficacy of 3-indole was also noted in various human esophageal squamous cell carcinoma cell lines. Furthermore, 3-indole caused most cancer cell lines, except for H1437, an accumulation in the G1 phase at a low concentration (10 μm), and increased in the sub-g1 region (an apoptosis indicator) at a high concentration (30 μm) at 24 h. Furthermore, the G2-M arrest by 48 h treatment with 30 μm 3-indole was also noted in H1437. The multi-effect of an anticancer drug concentration dependent on G1 or G2-M cell cycle arrest has also been shown for other compounds (Liebermann et al., 1995; Giannakakou et al., 2001; Blajeski et al., 2002). Cells respond to DNA damage by activating cell cycle checkpoints. p53 is one of the most commonly mutated genes found in human tumors (Friend 1994; Greenblatt et al., 1994). The function of p53 as a tumor suppressor has been demonstrated by experiments showing that the loss of p53 correlates with the loss of G1-S cell cycle transition regulation after DNA damage (Kastan et al., 1991; Park et al., 2001; Liu et al., 2003). The G2-M checkpoint induced by DNA damage can occur by either p53-dependent 37

52 or -independent through inhibition of RB phosphorylation mechanisms (Agarwal et al., 1995; Paules et al., 1995). Furthermore, the p21 protein at several sites in the cell cycle, targeting CDKs (4, 6, and 2), regulate cell cycle checkpoint can induces cell cycle arrest, is response to upregulation by p53 and by p53-independent mechanisms. In contrast to synthetic small-molecule compounds with an indole structure, such as vinorelbine, which induce almost complete G2-M arrest, 3-indole causes mainly G1 arrest. A number of anti-microtubule chemotherapy compounds characterized by the presence of an indole core nucleus have been obtained (Brancale & Silvestri 2007). Microtubules are crucial in G2-M phase and cell division (Jordan & Wilson 2004; Pellegrini & Budman 2005). The mechanism of action of many anti-microtubule drugs is interference with the normal formation of the mitotic spindle by either increasing microtubule depolymerization or tubulin polymerization leading to cell cycle arrest (Sorger et al., 1997). The different sensitivity of tumor and normal cells to anti-microtubule agents could possibly be due to (a) deficient function of G1 checkpoint (Trielli et al., 1996) and (b) deficiency of p53 tumor suppressor genes (Di Leonardo et al., 1997) in tumor cells. Our preliminary data shows that 3-indole induce the transient activation of p53 in early time and subsequently p21 increase expression in A549 or H1437 cells. Furthermore, the G2-M arrest by 48 h treatment with 30 μm 3-indole was also noted in H1437 cells (p53-mutation). 3-indole can induce G2 checkpoint protein cyclin B1 expression increases in H1435 cells (p53-mutation). Indeed, it has been found that breast 38

53 cancer cells with inactivated p53 failed to arrest in G2-M after paclitaxel treatment (Bacus et al., 2001). Therefore, we hypothesized that difference activation of p21 expression pathway is required for the induction of cell cycle arrest in 3-indole treated lung cancer cells with differ p53 status. Characterization of 3-indole-induced G2-M arrest in more cells with mutant p53 backgrounds with various treatment time of 3-indole is under investigation. In addition, microtubulin binding site of 3-indole will be further verified. DNA ladders appeared in various human lung cells in a time-dependent manner after 3-indole treatment. Apoptosis is a major control mechanism by which cells die if DNA damage is not repaired. Apoptosis occurs through two main pathways. The first pathway involves a member of the TNF receptor superfamily (extrinsic) and the second pathway involves the mitochondrial (intrinsic) pathway (Ghobrial et al., 2005). The Bcl-2 family of proteins constitutes a critical mediator in the mitochondrial pathway of apoptosis. Our results showed that treatment of A549 cells with 30 μm of 3-indole resulted in a time-dependent reduction in the levels of the anti-apoptotic Bcl-2 protein. Concomitantly, the level of pro-apoptotic Bax and Bad protein was increased. The decrease expression of anti-apoptotic Bcl-2 was also noted in a H1437 cells. Furthermore, the progression of apoptosis involves the activation of a cascade of proteases called caspases. Theoretically, the extrinsic pathway is related to the activation of caspase -8 and the intrinsic pathway is associated with activation of caspase -9. Both pathways converge to a common pathway involving the activation of caspase -3. As shown in our 39

54 data, 3-indole apparently stimulated caspases -3, caspase -9 and to a lesser extent caspase -8 activities in A549 cells. Together, these results suggested that 3-indole induced the execution of apoptosis through the activation of the intrinsic mitochondrial pathway. Various physical and chemical environmental stresses can activate apoptosis (Lavrik et al., 2005). One example of environmental stress-induced apoptosis is the loss of the MMP in cells and the subsequent induction of ROS by electron leakage from the mitochondrial electron transport chain. Various cancer cells have low-expression of some antioxidant enzymes (i. e. catalase and superoxide dismutase) (Ahmad et al., 2005), suggesting that induction of ROS in cancer cells may exhibit a potential target effect. Our data indicated that the MMP was decreased within min in A549 cells and a significant increase in ROS production was observed by 2 ~ 8 h in various human lung cancer cells. Furthermore, the ROS induced by 3-indole can be partially reduced by an inhibitor of mitochondrial respiratory chain complex I and a hydroxyl radical scavenger. Considerable evidence indicated that ROS, as signaling transduction molecules, induced apoptosis by the mitochondria pathway and DNA damage activation. Therefore, we hypothesized that 3-indole may cause DNA damage. PFGE analyses showed that 3-indole triggers DNA strand breaks in treated-a549 cells in a time-dependent manner and the triggered DNA damage can be partially recovered by incubation of 3-indole treated cells with ROS inhibitor. In addition, 3-indole-induced apoptosis can be rescued by co-treatment with ROS inhibitors. Together, these results suggested that oxidative stress may 40

55 potentially trigger 3-indole-induced DNA damage and may lead to apoptosis. ROS have been shown to induce various biological processes, including activation of the MAPK (Kamata et al., 2005; Gong et al., 2006). JNK-induced apoptosis has been shown to occur through the mitochondria (Tournier et al., 2000; Gong et al., 2006) and to require the presence of Bcl-2 family proteins (Donovan et al., 2002; Lei et al., 2002). Since ROS production occurred earlier than JNK activation, indicating that ROS may mediate the activation of JNK pathway and ultimately lead to cytotoxic effects such as DNA damage formation and apoptosis induced by 3-indole. Whether Bad induction occurs directly through JNK activation will be further tested as another mediator of 3-indole-induced apoptosis in NSCLC using a dominant negative and/or sirna approaches. JNK may induce expression of several apoptotic genes, such as Bcl-2 family proteins and Fas-L (Donovan et al., 2002; Lei et al., 2002; Su et al., 2005). The activation of protein phosphatase, PP2A, through the protein kinase C and/or phospholipase C pathways to eliminate phosphorylation on Jun protein (Avdi et al., 2002; Kong et al., 2004; Ray et al., 2005) is also worthy of further analysis. Activated Akt has numerous targets through a PI-3k-dependent pathway that are important regulators of the cell cycle, the apoptotic pathway, and the translational and transcriptional machinery. Furthermore, some studies have demonstrated that the inactivation of the pro-apoptosis protein, Bad, can also occur through the Ras/PI-3K/Akt pathway and that 41

56 the function of the PI-3K/Akt pathway is required to block apoptosis induced by oxidative stresses (Fernando & Wimalasena 2004; Zeng et al., 2006). In our pilot study, activation of Akt augmented the Bad protein after 3-indole treatment in A549 and CL1-1 cells. This involvement of the PI-3K/Akt pathway in Bad inactivation and the ROS stress pathway during 3-indole treatment is under investigation. Two different isoforms of cyclooxygenase (COX), also known as prostaglandin H synthase, catalyze the arachidonate metabolism, resulting in proinflammatory substances production, such as prostaglandins. One is COX-1, a housekeeping enzyme that is constitutively expressed, whereas the other is inducible COX-2. COX-1, which is expressed in almost all tissues, is important for maintenance of tissue homeostasis. In contrast, COX-2 is an inducible isozyme that is induced by cytokines, growth factors, and stresses at either transcriptional or post-transcriptional levels. Cytokines and growth factors, such as platelet derived growth factor and vascular endothelial growth factor, are known to promote angiogenesis (Koukourakis et al., 1997; Ohta et al., 1999). Angiogenesis is essential for tumor growth in vivo (Weidner & Folkman 1996; Thorpe 2004). COX-2 inhibition might be a strategy of chemopreventive and has been indicated to reduce the risk of colorectal, esophageal, gastric, lung, and breast cancers (Grubbs et al., 2000; Harris et al., 2000; Subbaramaiah & Dannenberg 2003). The p38 is activated by the cellular stresses, bacterial lipopolysaccharide, and inflammatory cytokines interleukin-1. The MAPK p38 has been shown to regulate COX-2 at both the transcriptional and post-transcriptional level (Lasa et al., 2001; Dean et al., 2003). In a 42

57 variety of cells treated with specific inhibitors of p38 block the accumulation of COX-2 mrna, suggesting a regulation of COX-2 gene expression by inhibition of p38. In our pilot study, decrease expression of p38 and COX-2 protein after 3-indole treatment in A549 cells. The data suggest that 3-indole is a potential COX-2 inhibitor and worth under investigation. Together our data indicated that that 3-indole is a multi-target inhibitor compound for cancer treatment. Further, cdna microarray data indicated that 3-indole affect other signaling pathways, such as Wnt signaling pathways. It is noteworthy that we also found that 3-indole caused reduction expression of the histone deacetylase 1 (HDAC1), which highly expresses and correlates with proliferative status in a variety of cancer cells (Minucci & Pelicci 2006; Wilson et al., 2006). HDAC1 has been identified as a potential target for cancer treatment (Acharya et al., 2005). Further studies on the role of HDAC1, upstream effectors of MAPK pathway, and other signaling pathways such as cell cycle control, angiogenesis, and metastasis during 3-indole induced apoptosis are under investigation. In the present study, we have shown for the first time that a novel synthetic indole structure compound, 3-indole, exhibits strong anti-tumor activities in both cell and animal models. 3-indole treatment induced remarkable tumor growth inhibition in treated animals. Toxicity in many tissues following chemotherapy is a major clinical concern (Iseri et al., 2007). The predominant organs for drugs and chemicals metabolism are liver and kidney (Anders 1980; Lee 2003). Therefore, we evaluated 43

58 serum biochemistry of liver and kidney functions in treated mice as a preliminary indicator of safety. Furthermore, evaluation of numerous histologic sections of tissues from animals bearing human A549 lung carcinoma tumor xenografts did not show any detectable pathologic abnormalities, as revealed by H&E staining. Importantly, at preclinical animal treated doses of 3-indole, no organ function damage in the liver or kidneys were observed in vivo. These data indicated that 3-indole is a potential to be tested as a lead pharmaceutical compound for cancer treatment. In conclusion, our data indicated that the 2-step synthetic 3-indole with high purity and yield induced intrinsic mitochondria pathway apoptosis in various lung cancer cells. In vivo anti-tumor activities against human xenografts support that 3-indole is a potential lead anti-cancer compound based on its strong tumor growth inhibition with favorable pharmacologic properties. In addition, 3-indole induced a time-dependent increase in ROS production and triggered DNA damage. The activation of JNK pathway induced by 3-indole may be mediated through ROS. Such multifactorial effect of an anti-cancer drug has also been shown for other indole compounds, such as I3C (Chinni & Sarkar 2002; Rahman et al., 2006). I3C has been shown to induce different enzymes activities in vitro and in vivo when given by different methods. The epiphenomenon suggests that 3-indole metabolic products in vivo, not the parent compound, represent the prerequisite for anticarcinogenesis (Dashwood et al., 1994). Since the cytotoxic dose of 3-indole used in the current study is relatively high compared to several anti-cancer drugs, we are 44

59 currently examining the cytotoxicity effect of 3-indole in cultured cells incubated with liver extract, which contains many metabolic enzymes. The preliminary data indicate a dramatic reduction of IC50 value of 3-indole in various lung cancer cell lines during co-incubation with liver extract. The result suggests that the effects of 3-indole appeared at 30 μm in our cell culture studies may be because 3-indole is a proximate anti-carcinogen which needs further metabolic activation to exhibit its full cytotoxic effect in cancer cell model. Characterization of in vivo metabolites of 3-indole is under the investigation. In addition, in vivo toxicity and pharmacological kinetic of 3-indole need to be further verified. 45

60 FIGURES A B Fig. 1. Chemical structure indole (A) and 1,1,3-tri(3-indolyl) cyclohexane (3-indole) (B). 46

61 cell survival (%) IMR90 H1299 H1435 CL1-1 H1437 A549 concentration (μm) Fig. 2. Cytotoxicity of 3-indole in normal human lung fibroblast cells IMR-90 and various human non-small cell lung carcinoma (NSCLC) cells (A549, H1299, H1435, CL1-1, and H1437). Cells were treated with various concentration 3-indole (0, 1, 2, 5, or 10 μm) for 24 h and cell proliferation was assessed by the MTT assay. 3-indole achieves an inhibitory concentration (IC) 50 value at ~ 10 μm in various human NSCLC cells. Data shown are the means of three independent experiments; bars, SE. 47

62 Fold O. D. increase fold increase of O. D A549 CL H1299 H H μm 2 μm 10 μm 30 μm Treatment time (h) 48

63 Fig. 3. In vitro proliferative effects of 3-indole in various human lung cancer cells (A549, H1299, H1435, CL1-1, and H1437 cells). Cells were treated with various concentration 3-indole (0, 2, 10, or 30 μm) at the indicated times for 24, 48, and 72 h and cell proliferation was assessed by the MTT assay. Y axis is the fold change of O. D. compared to that of untreated cells. Data shown are the means of three independent experiments; bars, SE. 49

64 A549 H1299 H1435 CL1-1 H indole 0 μm 3-indole 10 μm 3-indole 30 μm sub-g N 4N 2N 4N 2N 4N 2N 4N 2N 4N G1 arrest N 4N 2N 4N 100 2N 200 4N 2N 4N 2N 4N 0 2N 4N 2N 4N 2N 4N 2N 4N 2N 4N Fig indole induced cell cycle arrest and apoptosis in various human lung cancer cells. 3-indole-induced G1 arrest (indicated by arrows) and sub-g1 (indicated by arrow heads) in various human lung cancer cell lines (A549, H1299, H1435, CL1-1, and H1437). Cells were treated with various concentration 3-indole (0, 10, or 30 μm) at the indicated concentrations for 24 h. 50

65 indole 0 μm indole 30 μm G2 arrest N 4N 2N 4N Fig indole induced G2-M cell cycle arrest in H1437 lung cancer cells. H1437 cells were treated with various concentration 3-indole (0 or 30 μm) for 48 h. 3-indole blocks cell cycle at G2-M phase (indicated by arrow heads). Determination of cell cycle distribution was performed by FACScan flow cytometer. 51

66 A A549 (10 μm) 3-indole h p53 p21 GAPDH B H1437 (10 μm) (10 μm) 3-indole h P53 GAPDH 0 12 h P21 GAPDH C H1435 (10 μm) 3-indole h cyclin B1 GAPDH Fig. 6. Activation of the p53/p21 pathway is required for the induction of cell cycle arrest in 3-indole in various cells. (A). 3-indole-induced p53 and p21 increase expression in A549 cell lines. Cells were treated with 0 μm (0 h) or 10 μm 3-indole at the indicated times. (B). 3-indole-induced p53 and p21 increase expression in H1437 cell lines. Cells were treated with 0 μm (0 h) or 10 μm 3-indole at the indicated times for 0, 2, 4, 6, 8, and 12 h (left panel) or 12 h (right panel). (C). 3-indole-induced cyclin B1 increase expression in H1435 cell lines. Cells were treated with 0 μm (0 h) or 10 μm 3-indole at the indicated times. 52

67 24h 48h A549 H1299 H1435 CL1-1 A549 H indole 30 μm Fig indole induced DNA ladder appeared in various human lung cancer cells. Cell apoptosis assay using electrophoresis indicated that apoptotic DNA ladder appeared in various human lung cancer cells at h. Cells were treated with various concentration 3-indole (0 or 30 μm) at the indicated times for 24 and 48 h. ( symbol: 0 μm of 3-indole, + symbol: 30 μm of 3-indole). 53

68 A B A H h Bcl-2 Bax Bad caspase 9 active caspase 9 (cleaved form) caspase 3 active caspase 3 (cleaved form) caspase 8 GAPDH cytochrome C (cytosol) cytochrome C (mitochondria) h Bcl-2 Bad GAPDH 54

69 Fig. 8. Effects of 3-indole on the protein levels of Bcl-2, Bax, Bad, caspase-9, -3, -8 and cytochrome c in various lung cancer cells. (A). A549 cells. (B). H1437 cells. Preparation of cytosolic and mitochondria fractions was used for cytochrome c studies. Cells were treated with 0 μm (0 h) or 30 μm 3-indole at the indicated time for 4, 8, and 12 h in medium. Blotting experiments were repeated three times with similar results. 55

70 activity fold increase caspase 3 3 caspase 9 9 caspase h Treatment 3-indole (30μM) time Fig. 9. Induction of caspase activity by A549 cells. Cells were treated with 0 μm (0 h, untreated) or 30 μm 3-indole at the indicated times for 4, 8, 12, and 24 h in medium and lysed in caspase buffer. Enzymatic activities of caspase-3, -8, and -9 proteases were determined with fluorogenic substrates using corresponding caspase colorimetric assay kits. Fold induction in caspase activity was calculated as the ratio of the fluorescence of 3-indole-treated samples to that of untreated samples. Data shown are the means of three independent experiments; bars, SE. 56

71 Mitochondrial potential (%) min 0 μm 2 μm 10 μm 30 μm Treatment time Mitochondrial potential (%) μm 10 μm 30 μm 0 μm 10 μm 30 μm Treatment time h Fig indole induced that loss of the mitochondrial membrane potential (MMP) in A549 cells. A concentration-dependent change in mitochondrial membrane potential (MMP) was observed in min (upper panel). MMP dissipation can be detected up to 12 h post-treatment at 30 μm (lower panel). MMP was detected by DiOC6, which is a cationic fluorescent probe. Data shown are the means of three independent experiments; bars, SE. 57

72 0 μm 30 μm Fig indole induced that mitochondrial aggregates in A549 cells. Microscopic observations of A549 cells showed that cells treated with 3-indole (0 or 30 μm) for 12 h showed mitochondrial aggregates. 58

73 2 h ROS production (%) 60 0 μm 10 μm 30 μm A549 H1299 Fig indole induced the reactive oxygen species (ROS) production in various lung cancer cells. Cells were treated with 3-indole (0 or 10 μm) for 2 h. ROS production was measured using DCFH-DA as a fluorescent probe. Data shown are the means of three independent experiments; bars, SE. 59

74 ROS production (%) indole 3-indole / Rotenone control 3-indole 3-indole / Rotenone control treatment time (h) Fig. 13. ROS inhibitor reduced the 3-indole-induced ROS accumulation. Treated A549 cells with a ROS inhibitor rotenone (0.05 µm) effectively reduced the 3-indole-induced ROS accumulation. Cells were treated with 0 μm 3-indole (control, ), 30 μm 3-indole ( ) or co-treated with 3-indole (30 μm) and rotenone (0.05 µm) ( ). Data shown are the means of three independent experiments; bars, SE. 60

75 P< P<0.01 sub-g1 (%) indole (0 μm) 3-indole (30 μm) Rotenone (0.05 μm) NAC (5 mm) Fig. 14. ROS inhibitor reduced the 3-indole-induced apoptosis. Treated A549 cells with ROS inhibitor effectively reduced apoptosis during co-treatment with rotenone (lane 3) or NAC (lane 4) compared to 3-indole treatment alone (lane 2). Cells were treated with (+ symbol) or without ( symbol) 0 μm of 3-indole (control), 30 μm of 3-indole, rotenone (0.05 μm) or N-acetylcysteine (NAC) (5 mm). Data shown are the means of three independent experiments; bars, SE. 61

76 MW untreated UV (4J/cm 2 ) 3-indole (12h) 3-indole (24h) untreated UV (4J/cm 2 ) 3-indole (24h) 3-in + SP in + Rotenone MW Kb Kb Fig. 15. A time-dependent increase of DNA damage by 3-indole and it can be reversed by adding ROS inhibitor. A549 cells were treated with 0 μm 3-indole for 0 h (untreated), 4 J/cm 2 UV for 24 h, or 30 μm 3-indole for indicate times (12 and 24 h) (left panel). Untreated A549, or A549 cells treated with 4 J/cm 2 UV, 30 μm 3-indole, or co-treatment of 3-indole (30 µm) with ROS inhibitor rotenone (0.05 µm) or JNK inhibitor SP (20 µm) for 24 h are shown in right panel. Determination of DNA damage was performed by pulsed-field gel electrophoresis. 62

77 ratio (log2) cell cycle NM_ NM_ NM_ treatment time (h) ratio (log2) apoptosis NM_ NM_ NM_ treatment time (h) ratio (log2) 2 Wnt pathway NM_ NM_ treatment time (h) 63

78 ratio (log2) 2 NM_ Jak-STAT pathway treatment time (h) ratio (log2) 2 NM_ MAPK pathway treatment time (h) NM_ NM_ NM_ NM_ NM_ NM_ NM_ ratio (log2) 2 mtor pathway NM_ NM_ treatment time (h) 64

79 Fig. 16. Fold changes of specific genes in A549 cells treated with 3-indole. Changes in gene expression were presented as logarithmic ratios of fluorescence intensities between A549 RNA and universal human reference RNA. The number was converted to log-intensity log2 (The log-intensity log2r of the red dye of experimental A549 RNA versus the log-intensity log2g of the green dye of universal human reference RNA control.). The log intensity ratio is then calculated as expression value M= log 2 R/G. The genes selected were based on a 2-fold change of expression value. Cell cycle NM_002592, proliferating cell nuclear antigen (PCNA). NM_004964, histone deacetylase 1 (HDAC1). NM_007111, transcription factor Dp-1. Apoptosis NM_181869, apoptotic peptidase activating factor (APAF1) NM_004435, endonuclease G NM_000043, Fas (TNF receptor superfamily, member 6) Wnt signaling pathway NM_003502, AXIN 1 NM_177560, casein kinase 2 Jak-STAT signaling pathway NM_003999, oncostatin M receptor 65

80 MAPK signaling pathway NM_044472, cell division cycle 42 (CDC42) NM_002745, mitogen-activated protein kinase 1 (MAPK1) NM_001626, v-akt murine thymoma viral oncogene homolog 2 (AKT2) NM_002751, mitogen-activated protein kinase 11 (MAPK11, p38β) NM_004834, mitogen-activated protein kinase kinase kinase kinase 4 (MAPKKKK4, HGK) NM_003954, mitogen-activated protein kinase kinase kinase 14 (MAP3K14, NIK) NM_002752, mitogen-activated protein kinase 9 (MAPK9, JNK2) NM_002446, mitogen-activated protein kinase kinase kinase 10 (MAPKKK10) mtor signaling pathway NM_000618, insulin-like growth factor 1 (somatomedin C) NM_001968, eukaryotic translation initiation factor 4E (eif-4e) 66

81 A h P-ERK1 P-ERK2 ERK1 ERK2 P-p38 p38 P-JNK2 P-JNK1 JNK2 JNK1 P-c-Jun GAPDH B untreated 3-indole (12h) 3-in + SP in + Rotenone P-JNK2 P-JNK1 P-c-Jun GAPDH 67

82 Fig. 17. Activation of the JNK signaling pathways is required for the induction of apoptosis in 3-indole treated A549 cells. Cells were treated with 0 μm (0 h) or 30 μm 3-indole (4, 8, and 12 h) in medium. Effects of 3-indole on protein level of phosphorylated JNK, p38, ERK1/2 and c-jun expressions in A549 cells are shown in upper panel. Treatment A549 cells with 0 μm 3-indole for 0 h (untreated) and co-treatment with ROS inhibitor rotenone (0.05 µm) or JNK inhibitor SP (20 µm) reduced JNK and c-jun protein level compared to 3-indole (30 µm) treatment alone for 12 h (lower panel). 68

83 sub-g1 (%) indole (0 μm) 3-indole (30 μm) JNK I: SP (20 μm) ERK I: U0126 (10 μm) P< Fig. 18. JNK inhibitor (SP600125) reduced the 3-indole-induced apoptosis. Treated A549 cells with JNK inhibitor SP effectively reduced apoptosis compared to 3-indole treatment alone (Compare the data in lane 4 to that in lane 2). Cells were treated with (+ symbol) or without ( symbol) 0 μm of 3-indole (control), 30 μm of 3-indole, JNK inhibitor SP (20 μm) or ERK inhibitor U0126 (10 μm). Data shown are the means of three independent experiments; bars, SE. 69

84 A CL μm 3-indole h P-Akt B A μm 3-indole h P-Akt GAPDH C A μm 3-indole h COX-2 GAPDH Fig. 19. Effects of 3-indole on the protein levels of Akt (A, B) and COX-2 (C) in various lung cancer cells. A549 cells and CL1-1 cells were treated with 0 μμ 3-indole (0 h) or various concentration 3-indole (10, 30 μμ) in medium for indicate times then blotted with the corresponding antibodies indicated. 70

85 tumor size (mm 3 ) solvent 3-in 50mg/Kg 3-in 25mg/Kg A549 Days tumor size (mm 3 ) solvent 3-in 50mg/Kg H1435 Days Fig Indole effectively inhibited the growth of various human lung cancer cells (A549 and H1435) xenografts. A549 or H1435 cells (5 x 10 6 /100 μl) were injected s.c. into one flank per mice. Animals were treated i.p. with 3-indole (experiment final dose of 25 mg/kg or 50 mg/kg) or solvent (control). Tumor growth was examined after the volume of the tumor mass reached ~ 50 mm 3. 3-indole was dissolved in solvent [a vehicle mixture of DMSO/Cremophor EL/saline (2:1:7)]. 71

86 solvent (Liver) (Kidney) 20 X X 10 3-indole 50mg/Kg 20 X X 10 Fig. 21. H&E staining of A549 xenografts. H&E staining of paraffin embedded, 5 μm thick sections of the liver and kidney, from solvent and 3-indole-treated groups of mice (experiment final dose of 50mg/Kg) with A549 xenografts observed under 200X magnification. There were no apparent histopathologic differences in these tissues sections. 3-indole was dissolved in solvent [a vehicle mixture of DMSO/Cremophor EL/saline (2:1:7)]. 72

87 U / dl U / dl mg / dl GOT 0 30 GPT 0 Albumin mg / dl mg / / dl dl solvent 3-indole 50mg/Kg 0.0 Creatinine 0 BUN Fig. 22. serum biochemistry assays of A549 xenografts. Shows that 3-indole had no apparent change on serum biochemistry assays of liver and kidney functions in A549 xenograft model. Blood was obtained at the time of sacrifice. 3-indole was dissolved in solvent [a vehicle mixture of DMSO/Cremophor EL/saline (2:1:7)]. The organ functions tests included liver function tests, such as glutamic oxalacetic transaminase (GOT), glutamic pyvuvic transaminase (GPT), and albumin levels, and kidney function tests, such as blood urea nitrogen (BUN) and creatinine levels. 73

88 Appendix figure 1. In vitro proliferative effects of 3-indole in various esphogeal cancer cells (KYSE170, KYSE50, KYSE510, and KYSE70). Cells were treated with 0, 1, or 10 μm of 3-indole for 24 h and cell proliferation was assessed by the MTT assay. Data shown are the means of three independent experiments; bars, SE. 74

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101 BioFormosa (in press, 2008) APPENDIX A novel synthetic indole compound, 1,1,3-tri(3-indolyl)cyclohexane, inhibits microtubule polymerization in human lung cancer cells Ching-Hsiao Lee 1,2, Ching-Fa Yao 3, Guey-Jen Lee-Chen 1, Yi-Ching Wang 4 1. Department of Life Sciences, National Taiwan Normal University, Taipei, Taiwan 2. Department of Laboratory, Wei Gong Memorial Hospital, Miaoli, Taiwan 3. Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan 4. Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan Running Title: Antimicrotubule effect of 3-indole in lung cancer cell Grant supports: Supported in part by Grants NSC B MY3 and NSC M from the National Science Council (The Executive Yuan, Republic of China). Requests for reprints: Yi-Ching Wang, Ph.D.; Department of Pharmacology, National Cheng Kung University, No.1, University Road, Tainan 70101, Taiwan, R. O. C., TEL: ext.5502; FAX: ; ycw5798@mail.ncku.edu.tw 87

102 ABSTRACT BACKGROUND. Lung cancer is the most common malignancies in both men and women worldwide. Thus, the development of more effective anti-cancer drugs for lung cancer is urgently needed. METHODS. We generated a novel indole compound, 1,1,3-tri(3-indolyl)cyclohexane (3-indole), with high purity and in large quantities. 3-indole was tested for its biological activity in A549 and H1437 lung cancer cells. RESULTS. Our data indicated that 3-indole caused a concentration-dependent reduction in cell proliferation in human lung cancer cells but not in the normal lung cells. In addition, 3-indole induced G2-M cell cycle arrest in A549 and H1437 lung cancer cells to different extents. Using immunochemistry assay, the DMSO-treated control was shown to exhibit normal filamentous arrangement and organization of microtubule network whereas in A549 cells treated with 3-indole, almost complete loss of cellular microtubule networks throughout the cytoplasm was observed. Moreover, Western blot data showed that 3-indole dose-dependently inhibited microtubule polymerization in A549 cells. CONCLUSIONS. Based on its potent cell growth inhibition in lung cancer cell models, our data suggest that this novel synthetic 3-indole compound of high purity and yield is a potential antimicrotubule polymerization agent for cancer treatment. Key words: indole compound, lung cancer, tubule polymerization, and antimicrotubule. 88

103 INTRODUCTION Lung cancer is the most common malignancies in both men and women worldwide (Danesi et al., 2003; Jemal et al., 2007). Even with the recent advent of more effective molecular targeted therapies, the clinical responses to chemotherapy in patients with lung cancer are still unsatisfactory, with a 5-year overall survival in many countries generally less than 15% (Danesi et al., 2003). Thus, the development of effective anti-cancer drugs for lung cancer is urgently needed. Microtubules are main components of the cytoskeleton and are important for a variety of cell functions including maintenance of cell shape, transportation of vesicles, mitochondria and other components throughout cells, and segregation of chromosomes during cell division (Jordan and Wilson, 2004; Pellegrini and Budman, 2005). Microtubules are extremely dynamic polymers consisting of α tubulin and β-tubulin heterodimers arranged in the form of slender filamentous tubes that are constantly assembling (polymerization) or disassembling (depolymerization) (Jordan, 2002). Cancer cells acquire unlimited replication potential and continue to divide without progressing into immobility and senescence (Hayflick, 1997). The properties of uncontrolled proliferation and division make cancer cells extremely dependent upon the high dynamics of microtubule and hence sensitive to antimicrotubule compounds (Jordan and Wilson, 1998). Antimicrotubule agents (with various tubulin-binding sites), which have been found to interfere with tubulin/microtubules dynamic equilibrium, induce G2-M cell cycle arrest and trigger apoptosis (Woods et al., 1995; Jordan et al., 1996). These 89

104 findings indicate that microtubule is an important target for the development of novel anticancer drugs (Giannakakou et al., 2000). The clinically used antimicrotubule drugs generally fall into two main groups. One group includes vinca alkaloids, known as the microtubule-destabilizing agents such as vinorelbine, vincristine, and vinblastine. This type of agent inhibits microtubule polymerization and lead to the depolymerization of existing microtubules. The other group is known as the microtubule-stabilizing agents including taxanes, such as taxol (paclitaxel) and docetaxel, they stabilize microtubules and induce a net polymerization (Li and Sham, 2002). Despite the efficiency of antimicrotubule drugs in inhibiting the progression of some tumors, the important unsolved questions about the antitumor activities of antimicrotubule drugs concern the basis of their tissue specificities in many cancer types and the basis for the development of drug resistance to these agents usually occur during therapy (Gottesman, 2002). Several microtubule polymerization inhibitors, such as Vinca alkaloids, characterized by the presence of an indole core nucleus have been obtained from natural products or have been prepared by semi-synthesis (Brancale and Silvestri, 2007). We recently synthesized a novel indole compound, 1,1,3-tri(3-indolyl)cyclohexane (3-indole), with high purity and in large quantities (Ko et al., 2006). In the present study, we analyzed the biological activities especially the mechanisms involved in the anti-cancer growth activities of 3-indole in cell models. 3-indole induced G2-M cell cycle arrest in A549 and H1437 lung cancer cells to different extents. Furthermore, we found that cell 90

105 cycle arrest was induced via inhibition of microtubule polymerization in A549 cells. Together, these results indicated that 3-indole is a potential lead compound based on its antimicrotubule properties. 91

106 MATERIALS AND METHODS Cell Culture. Human non-small cell lung carcinoma cells (A549 and H1437) were maintained in DMEM. Media were supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin, and 100 g/ml of streptomycin (Invitrogen, Eugene, OR). The cells were maintained at 37 C in a humidified incubator containing 5% CO 2 in air. Cell Viability Assay. Cells were treated with DMSO or various concentrations of 3-indole (1, 5, or 10 μm) for 72 h. After treatment, the cells were washed with 1 PBS and then treated with 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in appropriate medium for 30 min at 37 C; cells generate a blue color when dissolved in DMSO. The intensity of the absorbance was measured using a reader for an enzyme-linked immunosorbent assay at a wavelength of 540 nm. Analysis of Cell Cycle Distribution. Cells were incubated in DMSO, 1 μm taxol (paclitaxel), 1 μm vinorelbine or various concentrations of 3-indole (10, 20, or 30 μm) for 24 h. Cells were collected by trypsinization, washed with 1 PBS, and fixed with ice-cold 80% ethanol at least overnight at -20 C until analysis. Fixed cells were collected by centrifugation, washed with 1 PBS, resuspended in 1 ml of 1 PBS containing 20 g/ml propidium iodide, 200 g/ml RNase A, and 0.1% triton X-100, and then incubated in the dark for 20 min. Determination of cell cycle distribution was performed by FACScan flow cytometer (BD, MountainView, CA) and calculated using ModFit LT software, version 2.0 (BD). 92

107 Immunocytochemistry. Cells were incubated in DMSO, 1 μm taxol, 1 μm vinorelbine or various concentrations of 3-indole (10, 20, or 30 μm) for 24 h. Cells were washed with 1 PBS, fixed with 1% formaldehyde for 20 min at room temperature, and washed twice with 1 PBST (1 PBS + 0.1% tween20) for 5 min. Cells were then incubated with 1 PBS containing primary antibodies α-tubulin (Cell Signaling Technologies, Beverly, MA) for 1 h at 37 o C. After washing with 1 PBS, cells were reincubated with FITC-conjugated secondary antibody (Upstate Biotechnology Inc., Lake Placid, NY) and DAPI (4'-6'-Diamidino-2-phenylindole, Sigma) in the dark room for 1 h at 37 o C. Cells then were washed with 1 PBS three times. Cellular microtubules were observed with an Olympus BX50 fluorescence microscope (Optical Elements Corporation, Dulles, VA). Western Blot Analysis. The assay was performed according to the method described by Juang et al. (Kuo et al., 2004). Cells were washed with 1 PBS before adding lysis buffer containing 20 mm Tris-HCl (ph 6.8), 1 mm MgCl 2, 2 mm EGTA, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm orthovanadate, and 0.5% Nonidet. Supernatants were collected after centrifugation at 13,000 rpm for 10 min at 4 C. The pellets were dissolved in an SDS-PAGE sampling loading buffer and heated at 95 C for 10 min. Cell lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk/1 PBST (1 PBS + 0.1% tween20) for 1 h at room temperature and probed with appropriate dilutions of primary antibody overnight at 4 C, as recommended by the manufacturers. The primary antibodies used were 93

108 α-tubulin (Cell Signaling Technologies, Beverly, MA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Novus Biologicals, Littleton, CO). Membranes were then washed three times with 1 PBST (1 PBS + 0.1% tween20) and subsequently incubated with appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After a further three washes with 1 PBST (1 PBS + 0.1% tween20), immunoreactive proteins were visualized using Western blot chemilluminescent reagents. 94

109 RESULTS 3-indole Inhibited the Growth of A549 lung cancer cells but not of normal lung cells. 3-indole is a novel, 2-step synthetic indole-like compound with high purity and yield. To test the cytotoxicity effect and future clinical use of 3-indole for anti-cancer treatment, IMR-90 normal human lung fibroblast cells and A549 human lung cancer cells were treated with 1, 5 or 10µM of 3-indole for 72 h and cell viability was assessed by the MTT assay. Fig. 1 shows that 3-indole caused a dose-dependent reduction in cell viability in A549 lung cancer cells. 3-indole achieved an IC50 value at 5 µm in A549 human lung cancer cells, whereas 3-indole did not show apparent cytotoxicity to the IMR-90 normal lung cells at this dose. 3-indole induced G2-M cell cycle arrest in A549 and H1437 cells. Indole-like compounds are known to arrest cells in G1 or G2/M, and substantially induce apoptosis (Brandi et al., 2003; Kuo et al., 2004). To determine whether the anti-cancer effect of 3-indole was associated with cell cycle deregulation, the cell cycle distribution was analyzed by flow cytometry. We studied whether G2-M cell cycle arrest could be induced in A549 cells treated with 10, 20 and 30 μm of 3-indole for 24 h. Flow cytometry results indicated that 10 μm of 3-indole caused A549 cancer cells to accumulate in G1 and partially G2-M cell cycles, and that a substantial increase in the sub-g1 region (an apoptosis indicator) resulted from treatment with 30 μm of 3-indole at 24 h (Figs. 2A-F). The G2-M arrest of 30 μm of 3-indole was also noted in H1437 lung cancer cells (Figs. 2G-H). These results indicate that 3-indole may induce cell death partly via G1 and 95

110 G2-M arrests. Effect of 3-indole on the cellular microtubule distribution in A549 cells. Microtubules are highly dynamic polymers composed of α tubulin and β-tubulin heterodimers that are constantly assembling (polymerization) or disassembling (depolymerization). Microtubules are crucial in G2-M phase and cell division. Antimicrotubule agents are known to arrest cells in G2-M, and substantially induce apoptosis (Giannakakou et al., 2000). To confirm that the partially G2-M arrest was caused by interference with tubulin/microtubules dynamic equilibrium, we employed immunocytochemistry to further examine the effect of 3-indole on cellular microtubule networks in A549 cells treated with1 μm taxol, 1 μm vinorelbine or various concentrations of 3-indole (10, 20, or 30 μm) treatment for 24 h. As shown in Fig. 3, the microtubule network exhibits normal filamentous arrangement and organization in A549 cells in the DMSO-treated control. However, 1 μm of vinorelbine caused cellular microtubule depolymerization as most cells had short microtubule fragments scattered throughout the cytoplasm. In contrast, 1 μm of taxol promoted microtubule polymerization with an increase in the density of cellular microtubules and formation of long thick microtubule bundles. Furthermore, 30 μm of 3-indole treatment resulted in findings similar to those of vinorelbine. We observed an almost complete loss of microtubules throughout the cytoplasm after 30 μm of 3-indole treatment. These results indicated that 3-indole may be an antimicrotubule polymerization agent. Validation of 3-indole-induced microtubule depolymerization by Western blot. 96

111 We thus used Western blot analysis to confirm the inhibition of microtubule polymerization by 3-indole. The effect of 3-indole on microtubule assembly was compared with those of taxol and vinorelbine. Inhibition of microtubule assembly was observed in A549 cells treated with 1 μm of vinorelbine. In contrast, 1 μm of taxol promoted tubulin polymerization. Similar to the effect of vinorelbine, 3-indole inhibited tubulin polymerization in a concentration-dependent manner (Fig. 4). Together, these results confirmed the antimicrotubule effect of 3-indole through inhibition of microtubule polymerization. 97

112 DISCUSSION In the present study, we show for the first time that a novel synthetic indole structure compound, 3-indole, exhibits anti-cancer growth activities and inhibits tubulin polymerization in cell model. 3-indole causes an accumulation in the G1 phase and partially increases in the G2-M phase in A549 human lung cancer cells. The G2-M arrest of 3-indole was also apparent in H1437 human lung cancer cells. Microtubules are crucial in G2-M phase and cell division (Jordan and Wilson, 2004; Pellegrini and Budman, 2005). The mechanism of action of many antimicrotubule drugs is interference with the normal formation of the mitotic spindle by either increasing microtubule depolymerization or tubulin polymerization leading to cell cycle arrest (Sorger et al., 1997). Our results show that treatment of A549 cells with 3-indole results in disruption of intracellular microtubule network as demonstrated in the immunocytochemistry studies. Furthermore, dose-dependent inhibition of tubulin polymerization by 3-indole in A549 cells is validated by Western blot assays. Together, these results suggest that 3-indole induces G2-M cell cycle arrest may be through the inhibition of microtubule polymerization. The different sensitivity of tumor and normal cells to antimicrotubule agents could possibly be due to (a) deficient function of G1 checkpoint (Trielli et al., 1996) and (b) deficiency of p53 tumor suppressor genes (Di Leonardo et al., 1997) in tumor cells. p53 is one of the most commonly mutated genes found in human tumors (Friend, 1994). The function of p53 as a tumor suppressor has been demonstrated by experiments showing 98

113 that the loss of p53 correlates with the loss of G1-S cell cycle transition regulation after DNA damage (Kastan et al., 1991; (Park et al., 2001; Liu et al., 2003). In contrast to synthetic small-molecule compounds with an indole structure, such as vinorelbine, which induce almost complete G2-M arrest, 3-indole causes different extents of G2-M arrest in various human lung cancer cells with different p53 statuses, including A549 (p53-wild) and H1437 (p53-mut). The multi-effect of an anti-cancer drug on G1 or G2/M cell cycle arrest has also been shown for other compounds (Blajeski et al., 2002). Characterization of 3-indole-induced G2-M arrest in more cells with null or mutant p53 backgrounds with various treatment time of 3-indole is under investigation. In addition, microtubulin binding site of 3-indole will be further verified. The tumor vasculature is a new target for cancer therapy. Tumor cells die rapidly unless they are supplied with oxygen and nutrients through the blood. Antimicrotubule compounds that bind to the colchicine or Vinca domain on microtubules, have undergone extensive development as antivascular agents, such as CA-4-P. CA-4-P induces cell death through rapid depolymerization of microtubules and formation of actin stress fibres with no evidence of apoptosis (Kanthou and Tozer, 2002). The difference between classical antimicrotubule agents and the novel vascular-targeting agents might be that the effects of potential vascular-targeting agents can (a) enter cells rapidly, (b) rapidly reverse its binding to tubulin or microtubules, (c) rapidly depolymerize microtubules, and (d) rapidly be metabolized or excreted (Tozer et al., 2002). Our preliminary data indicated that DNA damage induced by 3-indole can be rapidly reversed in cell model (Lee et al., 2008). Whether 3-indole might act as an antivascular agent is under investigation. 99

114 In conclusion, our data indicated that 3-indole, a novel synthetic indole compound, with high purity and yield, increases G2-M cells in A549 and H1437. In addition, 3-indole inhibits tubulin polymerization in A549 cells. Such effects of an anti-cancer drug have also been shown for other indole compounds, such as vinorelbine. Vinorelbine has been shown to affect different targets including tumor vasculature during cancer therapy. Characterization of 3-indole on various targets including vasculature of cancer cell is under investigation. 100

115 REFERENCES Blajeski, A. L., Phan, V. A., Kottke, T. J., and Kaufmann, S. H G1 and G2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells. J. Clin. Invest. 110: Brancale, A., and Silvestri, R Indole, a core nucleus for potent inhibitors of tubulin polymerization. Med. Res. Rev. 27: Brandi, G., Paiardini, M., Cervasi, B., Fiorucci, C., Filippone, P., De Marco, C., Zaffaroni, N., and Magnani, M A new indole-3-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6 expression, and induces G1 cell cycle arrest in both estrogen-dependent and estrogen-independent breast cancer cell lines. Cancer Res. 63: Danesi, R., de Braud, F., Fogli, S., de Pas, T. M., Di Paolo, A., Curigliano, G., and Del Tacca, M Pharmacogenetics of anticancer drug sensitivity in non-small cell lung cancer. Pharmacol. Rev. 55: Friend, S p53: a glimpse at the puppet behind the shadow play. Science. 265: Giannakakou, P., Sackett, D., and Fojo, T Tubulin/microtubules: still a promising target for new chemotherapeutic agents. J. Nati. Cancer Inst. 92: Gottesman, M. M Mechanisms of cancer drug resistance. Annu. Rev. Med. 53: Hayflick, L Mortality and immortality at the cellular level. A review. Biochemistry. 62:

116 Jordan, M. A Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr. Med. Chem. 2: Jordan, M. A., and Wilson, L Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr. Opin. Cell Biol. 10: Jordan, M. A., and Wilson, L Microtubules as a target for anticancer drugs. Nature Rev. 4: Ko, S., Lin, C., Tu, Z., Wang, Y. F., Wang, C. C., and Yao, C. F CAN and iodine-catalyzed reaction of indole or 1-methylindole with α,β-unsaturated ketone or aldehyde. Tetrahedron Lett. 47: Kuo, C. C., Hsieh, H. P., Pan, W. Y., Chen, C. P., Liou, J. P., Lee, S. J., Chang, Y. L., Chen, L. T., Chen, C. T., and Chang, J. Y BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res. 64: Lee, C. H., Yao, C. F., Huang, S. M., Ko, S., Tan, Y. H., and Wang, Y. C A novel two-step synthetic indole compound 1,1,3-tri(3-indolyl)cyclohexane inhibits cancer cell growth in lung cancer cells and xenograft models. Cancer. accepted. Li, Q., and Sham, H. L Discovery and development of antimitotic agents that inhibit tubulin polymerization for treatment of cancer. Exp. Opin. Ther. Pat. 12: Liu, Q., Hilsenbeck, S., and Gazitt, Y Arsenic trioxide-induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9 and synergy with APO2/TRAIL. Blood. 101: Park, J. W., Choi, Y. J., Jang, M. A., Baek, S. H., Lim, J. H., Passaniti, T., and Kwon, T. 102

117 K Arsenic trioxide induces G2/M growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in promonocytic U937 cells. Biochem. Biophys. Res. Commun. 286: Pellegrini, F., and Budman, D. R Review: tubulin function, action of antitubulin drugs, and new drug development. Cancer Invest. 23: Sorger, P. K., Dobles, M., Tournebize, R., and Hyman, A. A Coupling cell division and cell death to microtubule dynamics. Curr. Opin. Cell Biol. 9: Tozer, G. M., Kanthou, C., Parkins, C. S., and Hill, S. A The biology of the combretastatins as tumour vascular targeting agents. Int. J. Exp. Pathol. 83:

118 FIGURE LEGENDS cell survival (%) IMR90 A concentration (μm) Fig. 1. Viability assays of 3-indole in IMR-90 normal human lung cells and A549 human lung cancer cells. Cells were treated with 1, 5 or 10μM of 3-indole for 72 h and cell viability was assessed by the MTT assay. 104

119 105

120 Fig indole induced G2-M cell cycle arrest in A549 and H1437 lung cancer cells. A549 cells were treated with DMSO (A), 1 μm Taxol (B), 1 μm Vinorelbine (C), or 3-indole (10-30 μm) for 24 h (D-F), whereas H1437 cells were treated with DMSO or 3-indole (30 μm) for 24 h (G-H). 3-indole blocks cell cycle at G2-M phase (indicated by arrow heads) similar to that of known anti-microtubule agents (Taxol and Vinorelbine). Determination of cell cycle distribution was performed by FACScan flow cytometer. 106

121 Fig. 3. Effect of 3-indole on the cellular microtubule distribution in A549 cells. Cells were treated with DMSO (A), 1 μm Taxol (B), 1 μm Vinorelbine (C), or 30 μm 3-indole (D) for 24 h. The short depolymerized microtubules are presented in Vinorelbine- and 3-indole-treated cells, and the long polymerized microtubules are found in Taxol-treated cells. 107

122 Fig indole dose-dependently inhibits microtubule polymerization. A549 cells were treated with DMSO, 3-indole (10-30 μm), Vinorelbine, or Taxol for 24 h. Cell lysates were centrifuged to separate polymerized microtubules as described in Materials and Methods. 108

123 新穎的吲哚結構合成化合物 1,1,3-tri(3-indolyl)cyclohexane 抑制肺癌細胞株 microtubule 微管聚合作用探討 李慶孝 1,2, 姚清發 3, 李桂楨 1, 王憶卿 4* 1. 國立臺灣師範大學生命科學系 2. 苗栗財團法人為恭紀念醫院檢驗科 3. 國立臺灣師範大學化學系 4. 國立成功大學醫學院藥理所 摘要 目的 : 肺癌在世界各地無論男性或女性都是發病率 死亡率名列前茅的惡性腫瘤 因此, 發現與合成新穎的肺癌治療抗癌藥物是刻不容緩的工作 材料與方法 : 本研究團隊發展了一種新穎的吲哚結構合成化合物 1,1,3-tri(3-indolyl)cyclohexane (3-indole), 並藉由 A549 及 H1437 人類肺癌細胞株來探討新穎抗癌藥物對於肺癌細胞的毒殺作用及其機制 結果 : 新穎的抗癌藥物 3-indole 可以抑制 A549 和 H1437 肺癌細胞株細胞 109

124 生長並誘導細胞週期停滯在 G2-M 期 在 IMR90 正常肺細胞內,3-indole 無法抑制其細胞生長, 且細胞骨架 microtubule 微管為呈現網狀之完整分佈於整個細胞體內 ; 而經由免疫細胞染色法發現 3-indole 處理 A549 細胞後, 其 microtubule 微管網絡可見到幾乎受到破壞且聚集於細胞核周圍, 無法順利延伸散佈於整個細胞體內 此外, 西方墨點法分析顯示 3-indole 處理可抑制 A549 細胞 microtubule 微管聚合作用並呈現劑量相關性 結論 :3-indole 具有抑制 A549 和 H1437 肺癌細胞株細胞生長及抑制 A549 肺癌細胞株細胞骨架 microtubule 微管聚合作用, 顯示具有發展作為新穎的抗微管作用癌症用藥的價值 關鍵詞 : 吲哚結構化合物 肺癌 微管聚合作用 抗微管作用 * 通訊作者 : 王憶卿 (Yi-Ching Wang); ycw5798@mail.ncku.edu.tw;fax:

125 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 1 ID: jaganm Date: 3/6/08 Time: 20:36 Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR Novel 2-Step Synthetic Indole Compound 1,1,3-tri(3- Indolyl)Cyclohexane Inhibits Cancer Cell Growth in Lung Cancer Cells and Xenograft Models Ching-Hsiao Lee, MD 1,2 AQ1 Ching-Fa Yao, PhD 3 Sin-Ming Huang, MD 1 Shengkai Ko, MD 3 Yi-Hung Tan, MD 1 Guey-Jen Lee-Chen, MD 1 Yi-Ching Wang, PhD 4 1 Department of Life Sciences, National Taiwan Normal University, Taipei, Taiwan. 2 Department of Laboratory, Wei Gong Memorial Hospital, Miaoli, Taiwan. 3 Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan. 4 Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Supported in part by grants DOH96-TD-G , NSC B MY3, and NSC M from the National Science Council (Executive Yuan, Republic of China). Request for description of the preparation and chemical information for 1,1,3-tri(3-indolyl)cyclohexane (3-indole): Ching-Fa Yao, PhD, Department of Chemistry, National Taiwan Normal University, 88, Sec. 4, Tingchow Road, Taipei 11677, Taiwan, ROC. Fax: (011) ; cheyaocf@ntnu.edu.tw Address for reprints: Yi-Ching Wang, PhD, Department of Pharmacology, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan, ROC; Fax: (011) ; ycw5798@mail.ncku.edu.tw Received January 29, 2008; revision received March 21, 2008; accepted April 4, BACKGROUND. The clinical responses to chemotherapy in lung cancer patients are unsatisfactory. Thus, the development of more effective anticancer drugs for lung cancer is urgently needed. METHODS. A 2-step novel synthetic compound, referred to as 1,1,3-tri(3-indolyl)- cyclohexane (3-indole), was generated in high purity and yield. 3-Indole was tested for its biologic activity in A549, H1299, H1435, CL1-1, and H1437 lung cancer cells. Animal studies were also performed. RESULTS. The data indicate that 3-indole induced apoptosis in various lung cancer cells. Increased cytochrome-c release from mitochondria to cytosol, decreased expression of antiapoptotic Bcl-2, and increased expression of proapoptotic Bax were observed. In addition, 3-indole stimulated caspases-3, -9, and to a lesser extent caspase-8 activities in cancer cells, suggesting that the intrinsic mitochondria pathway was the potential mechanism involved in 3-indole-induced apoptosis. 3- Indole-induced a concentration-dependent mitochondrial membrane potential dissipation and an increase in reactive oxygen species (ROS) production. Activation of c-jun N-terminal kinase (JNK) and triggering of DNA damage were also apparent. Note that 3-indole-induced JNK activation and DNA damage can be partially suppressed by an ROS inhibitor. Apoptosis induced by 3-indole could be abrogated by ROS or JNK inhibitors, suggesting the importance of ROS and JNK stress-related pathways in 3-indole-induced apoptosis. Moreover, 3-indole showed in vivo antitumor activities against human xenografts in murine models. CONCLUSIONS. On the basis of its potent anticancer activity in cell and animal models, the data suggest that this 2-step synthetic 3-indole compound of high purity and yield is a potential candidate to be tested as a lead pharmaceutical compound for cancer treatment. Cancer 2008;000: Ó 2008 American Cancer Society. KEYWORDS: indole compound, lung cancer, mitochondria-mediated apoptosis, reactive oxygen species, c-jun N-terminal kinase. Lung cancer is the most frequent cause of cancer mortality in the world in both men and women. 1,2 Even with multimodality therapies and the recent advent of novel molecular targeted therapies (eg, epidermal growth factor receptor inhibitors), the clinical responses to chemotherapy in patients with lung cancer are still unsatisfactory, with a 5-year overall survival in many countries generally less than 15%. 1 Thus, the development of novel, more effective anticancer drugs for lung cancer is urgently needed. Natural and synthetic compounds with an indole structure have been shown to induce apoptosis through cell cycle arrest or a cellular stress activation mechanism. For example, Vinca alkaloids ª 2008 American Cancer Society DOI /cncr Published online 00 Month 2008 in Wiley InterScience (

126 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 2 ID: jaganm Date: 3/6/08 Time: 20:36 Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR CANCER Month 00, 2008 / Volume 00 / Number 0 and synthetic indole structure compounds, such as ABT-751 and BPR0L075, can cause G2/M arrest and apoptosis. 3-5 Indole-3-carbinol (I3C) and 3,3 0 -diindolylmethane, which are phytochemicals commonly found in cruciferous vegetables, induce G1 cell cycle arrest and apoptosis mediated by alterations in stress-activated protein kinase and activation of a DNA damage mechanism. 6-8 However, these natural and semisynthetic indole compounds have some disadvantages, such as harsh reaction conditions, long reaction times, and expensive preparation. We recently developed a novel 2-step synthesized indole compound, 1,1,3-tri(3-indolyl)cyclohexane (3- indole), in high purity and good yield. 9 In the present study we evaluated the biologic activities especially of the mechanisms involved in the anticancer growth activities of 3-indole in cell and animal models. 3- Indole induced G1 cell cycle arrest at low concentration (10 lm) and apoptosis at high concentration (30 lm) in various human lung cancer cell lines. Furthermore, we found that apoptosis was induced via an intrinsic mitochondrial pathway involving stress-activated pathways, including reactive oxygen species (ROS) and c-jun N-terminal kinase (JNK) activities. The events of apoptosis induced by 3-indole, such as mitochondrial membrane potential (MMP) dissipation, Bcl-2 inactivation, cytochrome-c release, and DNA ladder were observed. Moreover, in vivo antitumor activities against human xenografts in murine preclinical models indicated that 3-indole is a potential candidate to be tested as a lead pharmaceutical compound for cancer treatment. MATERIALS AND METHODS 3-Indole The compound 3-indole was synthesized at the Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan. 3-Indole was obtained as a solid powder in 90% yield. The detailed synthetic method was described in our previous article (Ko et al 9 ). Cell Culture Human nonsmall-cell lung cancer (NSCLC) cells (A549, H1299, H1437, and CL1-1) were maintained in DMEM and human NSCLC H1435 cells were maintained in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum. The cells were maintained at 378C in a humidified incubator containing 5% CO 2 in air. Cell Proliferation Assay Cells were treated with DMSO or various concentrations of 3-indole (2, 10, or 30 lm) for the indicated times. During the last 30 minutes of treatment the cells were treated with 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Cell proliferation was measured by the intensity of the absorbance at a wavelength of 540 nm. Analysis of Cell Cycle Distribution The assay was performed according to Kuo et al. 5 Cells were incubated with DMSO or various concentrations of 3-indole (10 or 30 lm) for 24 hours. Determination of cell cycle distribution was performed by FACScan flow cytometer (BD Bioscience, Mountain View, Calif). Determination of the Apoptotic DNA Ladder Fixed cells were collected by centrifugation, resuspended in 100 ll of DNA extraction buffer (0.2 M Na 2 HPO 4, 0.1 M citrate acid, and 0.5% Triton X-100, ph 7.8), and then incubated for 1 hour at 378C. After centrifugation the supernatant was collected and incubated with 5 ll RNase A (100 mg/ml) for 1 hour at 378C, followed by digestion with 5 ll proteinase K (20 mg/ml) for 1 hour at 378C. After electrophoresis the gels were stained and imaged. Evaluation of the Mitochondrial Transmembrane Potential The assay was basically performed according to the method described by Kuo et al. 5 Measurement of changes in MMP was performed using a FACScan flow cytometer (BD Bioscience). Western Blot Analysis Cell lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes. Membranes were blocked and probed with appropriate dilutions of primary antibody as recommended by the manufacturer. The primary antibodies used were caspase 3, caspase 8, caspase 9, and cytochrome-c (all from Upstate Biotechnology, Lake Placid, NY), Bcl-2, Bax (both from Chemicon, Temecula, Calif), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Novus Biologicals, Littleton, Colo). Membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were observed using Western blot chemiluminescent reagents. Determination of Caspase Activity Caspase activity was measured with the caspase colorimetric assay kit (BioVision, Mountain View, Calif) according to the manufacturer s instructions. After treatment, cells were lysed and the cell lysates were incubated with various synthetic caspase substrates (Ac-DEVD-pNA, Ac-LETD-pNA, and Ac-LEHD-pNA)

127 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 3 ID: jaganm Date: 3/6/08 Time: 20:36 Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR Apoptotic Mechanism of 3-Indole Treatment/Lee et al 3 to measure the activity of caspases-3, -8, and -9, respectively. After incubation at 378C the absorbance at 405 nm was measured. Immunocytochemistry The localization of mitochondria was detected using MitoTracker (Invitrogen, La Jolla, Calif) as a fluorescent probe. Cells were treated with DMSO or 30 lm 3-indole for the indicated times. During the last 30 minutes of treatment cells were treated with the MitoTracker (20 nm). Cellular mitochondria were observed with an Olympus BX50 fluorescence microscope (Optical Elements, Dulles, Va). Determination of Intracellular Reactive Oxygen Species The assay was performed as described by Juang et al. 10 Cells were treated with DMSO or 30 lm 3- indole for the indicated times. ROS production was measured using FACScan flow cytometer (BD Bioscience). Pulsed-Field Gel Electrophoresis The assay was performed according to the method described by Juang et al. 10 Cells were collected and resuspended in 1 3 PBS. PBS-suspended cells were mixed with 1% low-melting point agarose solution at a final concentration of cells per 0.1 ml of agarose block. The agarose plugs containing purified DNA were inserted into 1% agarose gels and the DNA was analyzed by pulsed-field gel electrophoresis using a FIGE Mapper Electrophoresis System (Bio- Rad, Hercules, Calif) for 16 hours at 128C. cdna Microarray Analysis A549 cells were treated with 30 lm 3-indole for 0, 4, 8, and 12 hours. Experimental A549 RNA was isolated using Trizol reagent (GIBCO BRL, Life Technology, Gaithersburg, Md). From each sample, total RNA (Control universal human reference RNA; Stratagene, La Jolla, Calif) and experimental A549 RNA were used to generate cdna. Microarray slides were scanned using GenePix 4000B Biochip Analyzer (Axon Instruments, Burlingame, Calif). Changes in gene expression were presented as logarithmic ratios of fluorescence intensities. The logarithmic ratios of each indicated time were then normalized for each gene to that of control RNA to obtain the expression pattern (the log-intensity log2r of the red dye vs the log-intensity log2g of the green dye, as well as the log intensity ratio M 5 log2r/g vs the log-intensity A 5 log2hrg, experimental Cy5 and control Cy3). The genes that showed substantial differences after drug treatment were selected based on at least a 2- fold change in expression. Subcutaneous Implantation of Cancer Cells in Animals and Monitoring of In Vivo Antitumoral Activity After Drug Treatments Athymic nu/nu female mice (ICR-Foxn1), 4 to 5 weeks of age, were obtained from the National Laboratory Animal Center (Republic of China, Taiwan). The animals were implanted subcutaneously with A549 or H1435 lung cancer cells in 0.1 ml Hanks balanced salt solution (HBSS) in 1 flank per mouse. The size of the tumor mass was measured and the tumor volume was calculated as 1/2 3 length 3 width 2 in mm 3. In human lung cancer xenograft studies, when tumors attained a mass of 50 mm 3, animals were treated intraperitoneally with 3-indole at 0.2 mg/day on Days 0, 2, 4, 6, and 8 (final dose, 50 mg/kg) or 0.1 mg/day on Days 0, 2, 4, 6, and 8 (final dose, 25 mg/kg), or a vehicle mixture control. A vehicle mixture contains alcohol / Cremophor EL / saline (2:1:7). The tumor size was measured after drug treatment. Before being sacrificed the animals were anesthetized and blood samples were collected by intracardiac puncture for the mice organ function test. Before organ dissection the animals were sacrificed by cervical dislocation. Tumor samples and mice organ tissues (including the lungs and kidneys) were resected, fixed with formalin and embedded in paraffin for histologic examination, stained with hematoxylin and eosin for microscopic evaluation, and examined by a pathologist. RESULTS 3-Indole Induced Cell Cycle Arrest and Apoptosis in Various Human Lung Cancer Cells 3-Indole is a novel, 2-step synthetic indole compound of high purity and yield. Its structure is shown in Figure 1A. To test the biologic activity of 3-indole for anticancer treatment, various human lung cancer cells including A549, H1299, H1435, CL1-1, and H1437 were treated with 2, 10, or 30 lm of 3-indole for the indicated times and cell proliferation was assessed by the MTT assay. Figure 1B shows that 3- indole caused a concentration-dependent reduction in cell proliferation with apparent inhibition of growth at low concentration (10 lm) and promotion of cell death at high concentration (30 lm) in various human lung cancer cells. To determine the cause of proliferation inhibition at low concentration (10 lm) and the promotion of cell death at high concentration (30 lm) of 3-indole we investigated whether cell cycle arrest and/or apoptosis could be induced in various human lung cancer cells (A549, H1299, H1435, CL1-1, and H1437 cells) treated with 3-indole at 10 and 30 lm for 24 hours. Flow cytometry AQ2 F1

128 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 4 ID: jaganm Date: 3/6/08 Time: 20:36 Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR CANCER Month 00, 2008 / Volume 00 / Number 0 indicated that 10 lm of 3-indole caused most cancer cell lines to accumulate in G1 phase and a substantial increase in the sub-g1 region (an apoptosis indicator) resulted from treatment with 30 lm of 3-indole at 24 hours (Fig. 1C). To confirm that the sub-g1 region was caused by apoptosis we performed a DNA ladder analysis and found that ladders appeared in various human NSCLC cells (A549, H1299, H1435, and CL1-1 cells) at 24 hours and in H1437 cells at 48 hours after 3-indole treatment (Fig. 1D). 3-Indole Induced Apoptosis Through the Activation of the Intrinsic Mitochondrial Pathway By using Western blot analysis to investigate the mechanism of 3-indole-induced apoptosis we found that treatment of A549 cells with 30 lm of 3-indole resulted in a time-dependent reduction in the levels of the antiapoptotic protein, Bcl-2. At the same time, the level of the proapoptotic protein, Bax, was concomitantly increased compared with the cells that were not treated with 3-indole (Fig. 2A). To further dissect the apoptosis pathway induced by 3-indole we performed Western blot analysis for cytochrome-c release and caspase protein expression and used different fluorogenic tetrapeptide substrates (Ac-DEVDpNA, Ac-LETD-pNA, and Ac-LEHD-pNA) to measure the activity of caspases-3, -8, and -9, respectively. 3- Indole increased the release of cytochrome-c from mitochondria to cytosol in 8 hours and stimulated caspases-3, -9 (an indicator of the intrinsic mitochondria pathway) and to a lesser extent caspase-8 (an indicator of the extrinsic membrane receptor pathway) activities in A549 cells (Fig. 2A,B). Together, these results showed that 3-indole induced the execution of apoptosis through the activation of the mitochondrial pathway. F2 FIGURE 1. 3-Indole-induced cell cycle arrest and apoptosis in various human lung cancer cells. (A) In vitro proliferative effects of 3-indole in various human lung cancer cells (A549, H1299, H1435, CL1-1, and H1437 cells). (B) Cells were treated with 2, 10, or 30 lm of 3-indole at the indicated times and cell proliferation was assessed by the MTT assay. Data shown are the means of 3 independent experiments; bars, SE. (C) 3-Indoleinduced G1 arrest (indicated by arrows) and sub-g1 (indicated by arrowheads) in various human lung cancer cell lines (A549, H1299, H1435, CL1-1, and H1437). Cells were treated with DMSO or 3-indole at the indicated concentrations for 24 hours. (D) Cell apoptosis assay using electrophoresis indicated that an apoptotic DNA ladder appeared in various human lung cancer cells at 24 to 48 hours. 3-Indole Induced Apoptosis by Reactive Oxygen Species Production and DNA Double-Strand Breaks in A549 Cells Several studies have shown that loss of MMP in cells triggers mitochondrial disruption and the generation of ROS. 6,11,12 ROSs are known to damage many molecules including proteins, RNA, and DNA. 13,14 We examined the changes in MMP and mitochondrial localization using DiOC6, a cationic fluorescent probe. A concentration-dependent change in MMP was observed in 15 to 30 minutes (Fig. 2C, upper left panel). The data in Figure 2C (upper right panel) shows that treatment with 10 or 30 lm of 3-indole decreased MMP in 4 hours, but only a high concentration (30 lm) of 3-indole continuously decreased MMP. Moreover, using cell fluorescence staining mitochondrial localization was detected by MitoTracker. In

129 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 5 ID: jaganm Date: 3/6/08 Time: 20:36 Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR Apoptotic Mechanism of 3-Indole Treatment/Lee et al 5 FIGURE 2. 3-Indole induced apoptosis through the activation of the intrinsic mitochondrial pathway. (A) Effects of 3-indole on the protein levels of Bcl-2, Bad, caspase-9, -3, -8, and cytochrome-c in A549 cells. Preparation of cytosolic and membrane fractions was used for cytochrome-c studies. Cells were treated with DMSO or 30 lm of 3-indole in medium for 0, 4, 8, and 12 hours. Blotting experiments were repeated 3 times with similar results. (B) Induction of caspase activity by A549 cells. Cells were treated with 30 lm 3-indole for the indicated times and lysed in caspase buffer. Enzymatic activities of caspase-3, -8, and -9 proteases were determined with fluorogenic substrates using corresponding caspase colorimetric assay kits. Fold induction in caspase activity was calculated as the ratio of the fluorescence of 3-indole-treated samples to that of untreated samples. Data shown are the means of 3 independent experiments; bars, SE. (C) A concentration-dependent change in mitochondrial membrane potential (MMP) was observed in 15 to 30 minutes (upper left panel). MMP dissipation can be detected up to 12 hours posttreatment at 30 lm (upper right panel). MMP was detected by DiOC6, which is a cationic fluorescent probe. Microscopic observations of A549 cells showed that cells treated with 30 lm 3-indole for 12 hours showed mitochondrial aggregates (lower right panel). FIGURE 3. 3-Indole induced apoptosis through the activation of the reactive oxygen species (ROS) production and DNA strand breaks. (A) 3-Indole-induced ROS production was observed in various human lung cancer cells (A549, H1435, and H1437) for 6 hours using DCFH-DA as a fluorescent probe. (B) Treated A549 cells with the ROS inhibitor rotenone (0.05 lm) effectively reduced the 3-indole-induced ROS accumulation. (C) Treated A549 cells with the ROS inhibitor rotenone effectively reduced the 3-indole-induced apoptosis. (D) A time-dependent increase of DNA damage by 3-indole using pulsed-field gel electrophoresis. Cotreatment of 3-indole with the ROS inhibitor rotenone (0.05 lm) or JNK inhibitor SP (20 lm) effectively reduced the 3-indole-induced DNA damage. AQ3 untreated cells mitochondria were evenly distributed in the cytoplasm. In 3-indole (30 lm)-treated A549 cells aggregated mitochondria increased after 12 hours and dendrite-like structures disappeared (Fig. 2C, lower panel). Next we examined the changes in ROS production and DNA damage in cells treated with 30 lm of 3-indole. A significant increase in ROS production was observed in various human NSCLC cells (A549, H1435, and H1437) at 6 hours (Fig. 3A). In addition, F3 A549 cells were treated with 3-indole and rotenone (0.05 lm, an inhibitor of mitochondrial respiratory chain complex I) or 3-indole and N-acetylcysteine (NAC) (5 mm, a hydroxyl radical scavenger). The results indicated a partial reversal of ROS production by rotenone (Fig. 3B) and reduced apoptosis during

130 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 6 ID: jaganm Date: 3/6/08 Time: 20:37 Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR CANCER Month 00, 2008 / Volume 00 / Number 0 TABLE 1 Fold Changes of Specific Genes in A549 Cells Treated With 3-indole (30 lm) AQ3 AQ4 A549 Genes 0h 4h 8h 12h Cell cycle, apoptosis NM_002592, proliferating cell nuclear antigen (PCNA) * 20.7* 21.4* NM_004964, histone deacetylase 1 (HDAC1) * 21.4* 20.9 NM_007111, transcription factor Dp * 21.1* 20.6* NM_181869, apoptotic peptidase activating factor (APAF1) : 0.9: 0.6: NM_004435, endonuclease G : 1.3: 1.1: NM_000043, Fas (TNF receptor superfamily, member 6) * 20.7* 21.0* Cell Communication, Kinase, cell signaling pathway NM_002273, keratin : 1.2: 1.0: NM_000224, keratin : 1.5: NM_044472, cell division cycle 42 (CDC42) : 1.3: 1.1: NM_002745, mitogen-activated protein kinase 1 (MAPK1) : 1.0: 1.0: NM_001626, v-akt murine thymoma viral oncogene homolog 2 (AKT2) * 21.5* 20.8* NM_002751, mitogen-activated protein kinase 11 (MAPK11, p38b) * 20.7* 20.7* NM_004834, mitogen-activated protein kinase kinase kinase kinase 4 (MAPKKKK4, HGK) * 21.0* 20.7* NM_003954, mitogen-activated protein kinase kinase kinase 14 (MAP3K14, NIK) * 21.2* 20.9* NM_002752, mitogen-activated protein kinase 9 (MAPK9, JNK2) : 0.5: 0.7: NM_002446, mitogen-activated protein kinase kinase kinase 10 (MAPKKK10) * 1.0* 0.5* NM_003999, oncostatin M receptor * 21.3* 20.1* NM_003502, AXIN * 21.1* 20.8* NM_177560, casein kinase * 21.6* 21.6* NM_000618, insulin-like growth factor 1 (somatomedin C) * 20.7* 21.2* NM_001968, eukaryotic translation initiation factor 4E (eif-4e) * 21.4* 20.4* The number for indicated 3-indole treatment time is the gene expression levels of samples compared with 0h. The data were converted to log-intensity log2. The genes selected were based on a 2-fold change of expression value. *Decreased expression compared to the 0h. :Increased expression compared with the 0h. cotreatment with rotenone or NAC compared with 3- indole treatment alone (Fig. 3C). Because there was an increase in ROS production we decided to assess the degree of DNA strand break damage using pulsed-field gel electrophoresis (PFGE). A549 cells, after 30 lm 3-indole treatment, exhibited a change in DNA damage at 24 hours (Fig. 3D, left panel). In addition, we treated A549 cells with both 3-indole and rotenone (0.05 lm). The data indicated that cotreatment with rotenone reduced DNA damage compared with 3-indole treatment alone (Fig. 3D, right panel). cdna Microarray Analysis to Search for Differential Expressed Genes After 3-Indole Treatment To reveal more potential targets and pathways involved in 3-indole treatment we performed cdna microarray analysis on A549 cells treated with 30 lm of 3-indole and harvested the RNA at 0, 4, 8, and 12 hours. The dose chosen was close to the dose needed for apoptosis induction. The rationale for the indicated times was to capture the expression profiles of genes that were involved in the apoptotic processes. We found many differentially expressed genes that are related to cell cycle, apoptosis, and cell-signaling pathways (Table 1). For example, we found that 30 lm of 3-indole caused changes in the mrna levels of several mitogen-activated protein kinase (MAPK) signaling proteins such as p38b and JNK2. Activation of the JNK Signaling Pathways Is Required for the Induction of Apoptosis in 3-Indole-Treated A549 Cells cdna microarray data revealed that expression of several proteins in the MAPK pathway changed after 3-indole treatment. In addition, ROS has been shown to induce various biologic processes, including activation of the MAPK pathway. 6,15 Therefore, we performed Western blot to confirm whether the MAPK signaling pathway was activated after 3-indole treatment and whether ROS was involved in 3-indoleinduced MAPK activation. Cell lysates were subjected to Western blot analysis using antiphospho-mapk antibodies (ERK1 of 2, JNK, and p38) to detect phosphorylated activated MAPK family proteins. The data in the left panel of Figure 4A shows that 3- indole increased the activation of JNK1 in 4 hours T1 F4

131 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 7 ID: jaganm Date: 3/6/08 Time: 20:37 Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR Apoptotic Mechanism of 3-Indole Treatment/Lee et al 7 FIGURE 4. Activation of the JNK signaling pathways is required for the induction of apoptosis in 3-indole-treated A549 cells. (A) Effects of 3-indole on phosphorylated JNK, p38, ERK1 of 2, and c-jun expressions in A549 cells (left panel). Cotreatment A549 cells with the ROS inhibitor rotenone (0.05 lm) or JNK inhibitor (SP600125, 20 lm) reduced JNK and c-jun protein expression compared with 3-indole (30 lm) treatment alone (right panel). Experiments were repeated 3 times with similar results. (B) Effects of JNK inhibitor (SP600125) and ERK inhibitor (U0126) on cell cycle distribution after treatment with (plus symbol) or without (minus symbol) 30 lm of 3-indole. The change of sub-g1 phase is indicated by arrows in cotreatment of JNK inhibitor and 3-indole compared with the 3-indole treatment alone. and JNK2 in 8 hours. In addition, we found that 3- indole increased the phosphorylation of c-jun, a major nuclear factor of the MAPK signaling pathway, in 4 to 12 hours. Furthermore, we cotreated A549 cells with rotenone (0.05 lm), SP (20 lm, an inhibitor of JNK), or U0126 (10 lm, an inhibitor of ERK). The results indicated that cotreatment of 3- indole with rotenone or SP reduced activated JNK and c-jun protein expression compared with 3- indole treatment alone (Fig. 4A, right panel). The data in Figure 4B shows that treatment of A549 cells with a combination of the JNK inhibitor and 3-indole caused a significant reduction in 3-indole-induced apoptosis when compared with the cells treated with 3-indole alone, whereas no effect of ERK inhibitor on 3-indole-induced apoptosis was seen. In addition, cotreatment with 3-indole and SP reduced DNA damage compared with 3-indole treatment alone (Fig. 3D, right panel). The results indicated that inhibition of JNK activation protects against the cytotoxic effects of 3-indole and that ROS may play a role in JNK activation. 3-Indole Effectively Inhibited the Growth of Human A549 and H1435 Xenografts To examine whether 3-indole treatment inhibited A549 cell growth in vivo we followed the tumor growth in 3-indole and vehicle-treated animals (ICR- Foxn1). Solvent control cells were treated with a vehicle mixture of alcohol / Cremophor EL / saline (2:1:7). To further determine the effect of 3-indole over an extended treatment period, tumor size was measured in each animal. In the meantime, 3- indole-treated animals were sacrificed and processed

132 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 8 ID: jaganm Date: 3/6/08 Time: 20:37 Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR CANCER Month 00, 2008 / Volume 00 / Number 0 C O L O R FIGURE 5. 3-Indole effectively inhibited the growth of various human lung cancer cells (A549 and H1435) xenografts. (A) A549 (left panel) or H1435 (right panel) cells ( /100 ll) were injected subcutaneously into 1 flank per mice. Animals were treated intraperitoneally with 3-indole (final dose of 25 mg/kg or 50 mg/kg) or a vehicle mixture control. Tumor growth was examined after the volume of the tumor mass reached 50 mm 3. (B) H&E staining of paraffinembedded, 5-lm thick sections of the liver and kidney from untreated and 3-indole-treated groups of mice with A549 xenografts observed under 2003 magnification. There were no apparent histopathologic differences in these tissues sections. (C) 3-Indole had no apparent change on serum biochemistry assays of liver and kidney functions in A549 xenograft model. Blood was obtained at the time of sacrifice. F5 for evaluation of any possible changes in histopathology and serum biochemistry. Figure 5A shows the tumor growth in control vehicle-treated animals compared with 3-indole treatments. Treatment with 3-indole (25 or 50 mg/kg intraperitoneally) resulted in tumor growth inhibition compared with that produced by control vehicletreated animals bearing A549 cell xenografts (Fig. 5A, left panel). The same observations were also noted in an H1435 cell xenograft model (Fig. 5A right panel). Evaluation of numerous histologic sections of these tissues from animals bearing human A549 xenografts did not indicate any detectable pathologic abnormalities, as examined by H&E staining (Fig. 5B). In addition, 3-indole therapy caused no detectable toxicity on tissues and did not affect organ functions. The organ function tests included liver function tests, such as glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and albumin levels, and renal function tests, such as blood urea nitrogen (BUN) and creatinine levels. The organ functions were similar between the 3-indole-treated and the vehicle-treated groups (Fig. 5C). DISCUSSION We evaluated the biologic activities, especially the mechanisms, involved in the anticancer growth of 3- indole in cell and animal models. 3-Indole caused

133 J_ID: Z7B Customer A_ID: C Cadmus Art: CNCR23619 Date: 3-JUNE-08 Stage: I Page: 9 Apoptotic Mechanism of 3-Indole Treatment/Lee et al 9 C O L O R FIGURE 6. l l l an accumulation in the G1 phase at a low concentration (10 lm), and increased in the sub-g1 region (an apoptosis indicator) in all lung cancer cell lines at a high concentration (30 lm). DNA ladders appeared in various human lung cells in a time-de- ID: jaganm Date: 3/6/08 Time: 20:37 pendent manner after 3-indole treatment. Apoptosis occurs through 2 main pathways. The first pathway involves a member of the TNF receptor superfamily (extrinsic) and the second pathway involves the mitochondrial (intrinsic) pathway.16 The Bcl-2 family of Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR AQ3