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)APPENDIX BioFormosa (in press, 2008). A novel synthetic indole compound, 1,1,3-tri(3-indolyl)cyclohexane, inhibits microtubule polymerization in human lung cancer cells. Ching-Hsiao Lee1,2, Ching-Fa Yao3, Guey-Jen Lee-Chen1, Yi-Ching Wang4. 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 NSC96-2628-B-006-048-MY3 and NSC96-2627-M-006-005 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: +886-6-2353535 ext.5502; FAX: +886-6-2749296; E-mail: [email protected]. 87.

(2) 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.

(3) 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.

(4) 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.

(5) 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.

(6) 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% CO2 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.

(7) 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 37oC. 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 37oC. 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 MgCl2, 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.

(8) 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.

(9) 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.

(10) 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.

(11) 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.

(12) 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.

(13) 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.

(14) 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.

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(18) cell survival (%). FIGURE LEGENDS. 100. 50 IMR90 A549 0 0. 5. 10. 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.

(19) 105.

(20) Fig. 2. 3-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.

(21) 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.

(22) Fig. 4. 3-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.

(23) 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.. 2.. 國立臺灣師範大學生命科學系苗栗財團法人為恭紀念醫院檢驗科 3.. 4.. 國立臺灣師範大學化學系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.

(24) 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)； E-mail: [email protected]；FAX：886-6-2749296. 110.

(25) J_ID: Z7B Customer A_ID: C2674-07. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 1. 1. Novel 2-Step Synthetic Indole Compound 1,1,3-tri(3Indolyl)Cyclohexane Inhibits Cancer Cell Growth in Lung Cancer Cells and Xenograft Models. AQ1. Ching-Hsiao Lee, MD1,2 Ching-Fa Yao, PhD3 Sin-Ming Huang, MD1 Shengkai Ko, MD3 Yi-Hung Tan, MD1 Guey-Jen Lee-Chen, MD1 Yi-Ching Wang, PhD4. 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 1. cells. Increased cytochrome-c release from mitochondria to cytosol, decreased. Department of Life Sciences, National Taiwan Normal University, Taipei, Taiwan.. expression of antiapoptotic Bcl-2, and increased expression of proapoptotic Bax. 2. were observed. In addition, 3-indole stimulated caspases-3, -9, and to a lesser extent. 3. caspase-8 activities in cancer cells, suggesting that the intrinsic mitochondria pathway was the potential mechanism involved in 3-indole-induced apoptosis. 3-. Department of Laboratory, Wei Gong Memorial Hospital, Miaoli, Taiwan. Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan.. Indole-induced a concentration-dependent mitochondrial membrane potential dissipation and an increase in reactive oxygen species (ROS) production. Activation. 4. Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.. 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 Supported in part by grants DOH96-TD-G-111004, NSC96-2628-B-006-048-MY3, and NSC962627-M-006-005 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) 886-2-29324249; E-mail: [email protected] Address for reprints: Yi-Ching Wang, PhD, Department of Pharmacology, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan, ROC; Fax: (011) 886-6-2749296; E-mail: [email protected] Received January 29, 2008; revision received March 21, 2008; accepted April 4, 2008.. compound for cancer treatment. Cancer 2008;000:000–000. 2008 American Cancer Society.. KEYWORDS: indole compound, lung cancer, mitochondria-mediated apoptosis, reactive oxygen species, c-Jun N-terminal kinase.. L. ung 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 10.1002/cncr.23619 Published online 00 Month 2008 in Wiley InterScience (www.interscience.wiley.com).. ID: jaganm. Date: 3/6/08. Time: 20:36. Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373.

(26) J_ID: Z7B Customer A_ID: C2674-07. 2. CANCER. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 2. 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 3,30 -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 (3indole), 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. 3Indole 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 al9). 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% CO2 in air. Cell Proliferation Assay Cells were treated with DMSO or various concentrations of 3-indole (2, 10, or 30 lM) for the indicated. ID: jaganm. Date: 3/6/08. Time: 20:36. 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 Na2HPO4, 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). Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373.

(27) J_ID: Z7B Customer A_ID: C2674-07. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 3. Apoptotic Mechanism of 3-Indole Treatment/Lee et al. 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 3indole 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 1 3 106 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 (BioRad, 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 2fold change in expression.. ID: jaganm. Date: 3/6/08. Time: 20:36. 3. 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 5 3 106 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 width2 in mm3. In human lung cancer xenograft studies, when tumors attained a mass of 50 mm3, 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.. AQ2. 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 3indole 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. Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373. F1.

(28) J_ID: Z7B Customer A_ID: C2674-07. 4. CANCER. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 4. 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. 3Indole 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.. 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.. ID: jaganm. Date: 3/6/08. Time: 20:36. 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. Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373. F2.

(29) J_ID: Z7B Customer A_ID: C2674-07. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 5. 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. AQ3. 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 SP600125 (20 lM) effectively reduced the 3-indole-induced DNA damage.. 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. ID: jaganm. Date: 3/6/08. Time: 20:36. was observed in various human NSCLC cells (A549, H1435, and H1437) at 6 hours (Fig. 3A). In addition, 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. Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373. F3.

(30) J_ID: Z7B Customer A_ID: C2674-07. 6. CANCER. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 6. 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 Cell cycle, apoptosis NM_002592, proliferating cell nuclear antigen (PCNA) NM_004964, histone deacetylase 1 (HDAC1) NM_007111, transcription factor Dp-1 NM_181869, apoptotic peptidase activating factor (APAF1) NM_004435, endonuclease G NM_000043, Fas (TNF receptor superfamily, member 6) Cell Communication, Kinase, cell signaling pathway NM_002273, keratin 8 NM_000224, keratin 18 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, p38b) 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) NM_003999, oncostatin M receptor NM_003502, AXIN 1 NM_177560, casein kinase 2 NM_000618, insulin-like growth factor 1 (somatomedin C) NM_001968, eukaryotic translation initiation factor 4E (eIF-4E). 0h. 4h. 8h. 12h. 0.9 20.9 20.2 20.2 0.8 20.1. 0.4* 21.6* 21.1* 1.2: 1.3: 20.2*. 20.7* 21.4* 21.1* 0.9: 1.3: 20.7*. 21.4* 20.9 20.6* 0.6: 1.1: 21.0*. 0.8 1.4 0.3 20.2 0 0.3 20.2 20.7 0.1 1.1 0.4 20.3 20.9 20.6 0.5. 1.1: 1.4 1.3: 0.1: 20.3* 20.3* 20.9* 20.8* 0.2: 0.8* 20.4* 20.8* 21.3* 20.7* 20.1*. 1.2: 1.6: 1.3: 1.0: 21.5* 20.7* 21.0* 21.2* 0.5: 1.0* 21.3* 21.1* 21.6* 20.7* 21.4*. 1.0: 1.5: 1.1: 1.0: 20.8* 20.7* 20.7* 20.9* 0.7: 0.5* 20.1* 20.8* 21.6* 21.2* 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 3indole 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.. ID: jaganm. Date: 3/6/08. Time: 20:37. 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 3indole increased the activation of JNK1 in 4 hours. Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373. T1. F4.

(31) J_ID: Z7B Customer A_ID: C2674-07. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 7. 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 3indole 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), SP600125 (20 lM, an inhibitor of JNK), or U0126 (10 lM, an inhibitor of ERK). The results indicated that cotreatment of 3indole with rotenone or SP600125 reduced activated JNK and c-Jun protein expression compared with 3indole 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 SP600125 reduced. ID: jaganm. Date: 3/6/08. Time: 20:37. 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 (ICRFoxn1). 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, 3indole-treated animals were sacrificed and processed. Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373.

(32) J_ID: Z7B Customer A_ID: C2674-07. 8. CANCER. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 8. 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 (5 3 106/100 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 mm3. (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 addi-. ID: jaganm. Date: 3/6/08. Time: 20:37. tion, 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 3indole in cell and animal models. 3-Indole caused. Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373.

(33) J_ID: Z7B Customer A_ID: C2674-07. 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/C2CNCR080373. AQ3.

(34) J_ID: Z7B Customer A_ID: C2674-07. 10. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 10. CANCER Month 00, 2008 / Volume 00 / Number 0. proteins constitutes a critical mediator in the mitochondrial pathway of apoptosis.16 Our results showed that treatment of A549 cells with 30 lM of 3-indole resulted in a time-dependent reduction in the levels of the antiapoptotic Bcl-2 protein. Concomitantly, the level of proapoptotic Bax protein was increased. 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 data, 3-indole apparently stimulated caspases-3, caspase-9 and to a lesser extent caspase8 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.17 One example of environmental stress-induced apoptosis is the loss of 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 (ie, catalase and superoxide dismutase),18 suggesting that induction of ROS in cancer cells may exhibit a potential target effect. Our data indicated that MMP was decreased within 4 hours in A549 cells and a significant increase in ROS production was observed by 6 to 8 hours 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 an ROS inhibitor. In addition, 3-indoleinduced apoptosis can be rescued by cotreatment with ROS inhibitors. Together, these results suggested that oxidative stress may potentially trigger 3-indoleinduced DNA damage and may lead to apoptosis. ROS have been shown to induce various biological processes, including activation of MAPK.6,15 JNKinduced apoptosis has been shown to occur through the mitochondria6,19 and to require the presence of Bcl-2 family proteins.20,21 Our data indicated that ROS may mediate the activation of the JNK pathway. ID: jaganm. Date: 3/6/08. Time: 20:38. and ultimately lead to cytotoxic effects such as DNA damage formation and apoptosis induced by 3indole. Furthermore, cDNA microarray data indicated that 3-indole affect other signaling pathways, such as PI3K-Akt and Wnt signaling pathways. It is noteworthy that we also found that 3-indole caused a reduction in expression of the histone deacetylase 1 (HDAC1), which highly expresses and correlates with proliferative status in a variety of cancer cells.22,23 HDAC1 has been identified as a potential target for cancer treatment.24 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-indoleinduced 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 antitumor activities in both cell and animal models. 3-Indole treatment induced remarkable tumor growth inhibition in treated animals. Toxicity in many tissues after chemotherapy is a major clinical concern.25 The predominant organs for drugs and chemicals metabolism are liver and kidney.26,27 Therefore, we evaluated 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 3indole 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 antitumor activities against human xenografts support that 3-indole is a potential lead anticancer 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 the JNK pathway induced by 3-indole may be mediated through ROS. Such a multifactorial effect of an anticancer drug has also been shown for other indole compounds, such as I3C.28,29 I3C has been shown to induce different enzyme 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.30 Because the cytotoxic dose of 3-indole. Path: J:/Production/CNCR/Vol00000/080373/3B2/C2CNCR080373.

(35) J_ID: Z7B Customer A_ID: C2674-07. Cadmus Art: CNCR23619. Date: 3-JUNE-08. Stage: I. Page: 11. Apoptotic Mechanism of 3-Indole Treatment/Lee et al. used in the current study is relatively high compared with several anticancer drugs, we are 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 the IC50 value of 3indole in various lung cancer cell lines during coincubation with liver extract. The result suggests that the effects of 3-indole that appeared at 30 lM in our cell culture studies may be because 3-indole is a proximate anticarcinogen that needs further metabolic activation to exhibit its full cytotoxic effect in a cancer cell model. Characterization of in vivo metabolites of 3-indole is under the investigation. In addition, in vivo toxicity and the pharmacologic kinetics of 3-indole need to be further verified.. 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