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
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
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,MD1,2
AQ1 Ching-Fa Yao,PhD3
Sin-Ming Huang,MD1
Shengkai Ko,MD3
Yi-Hung Tan,MD1
Guey-Jen Lee-Chen,MD1
Yi-Ching Wang,PhD4
1Department of Life Sciences, National Taiwan Normal University, Taipei, Taiwan.
2Department of Laboratory, Wei Gong Memorial Hospital, Miaoli, Taiwan.
3Department of Chemistry, National Taiwan Nor-mal University, Taipei, Taiwan.
4Department of Pharmacology, College of Medi-cine, National Cheng Kung University, Tainan, Tai-wan.
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 can-cer 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 path-way was the potential mechanism involved in 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 sup-pressed 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 antitu-mor 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;000:000–000. 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 thera-pies (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 gen-erally less than 15%.1 Thus, the development of novel, more effec-tive 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
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Supported in part by grants DOH96-TD-G-111-004, 2628-B-006-048-MY3, and NSC96-2627-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)cyclo-hexane (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.
ª2008 American Cancer Society
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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 -diindo-lylmethane, 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 dis-advantages, such as harsh reaction conditions, long reaction times, and expensive preparation.
We recently developed a novel 2-step synthesized indole compound, 1,1,3-triindolyl)cyclohexane (3-indole), in high purity and good yield.9In the present study we evaluated the biologic activities especially of the mechanisms involved in the anticancer growth activities of 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. Further-more, we found that apoptosis was induced via an intrinsic mitochondrial pathway involving stress-acti-vated 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) dissipa-tion, Bcl-2 inactivadissipa-tion, cytochrome-c release, and DNA ladder were observed. Moreover, in vivo antitu-mor activities against human xenografts in murine preclinical models indicated that 3-indole is a poten-tial 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 in90% 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 main-tained in RPMI 1640 medium. All media were sup-plemented with 10% fetal bovine serum. The cells were maintained at 378C in a humidified incubator containing 5% CO2in air.
Cell Proliferation Assay
Cells were treated with DMSO or various concentra-tions 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-dimethylthia-zol-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 concen-trations of 3-indole (10 or 30 lM) for 24 hours.
Determination of cell cycle distribution was per-formed by FACScan flow cytometer (BD Bioscience, Mountain View, Calif).
Determination of the Apoptotic DNA Ladder
Fixed cells were collected by centrifugation, resus-pended 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 pro-teinase K (20 mg/mL) for 1 hour at 378C. After elec-trophoresis 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 electro-phoretically 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 de-hydrogenase (GAPDH; Novus Biologicals, Littleton, Colo). Membranes were then incubated with appro-priate 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 col-orimetric 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)
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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 fluores-cent 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 micro-scope (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 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 (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 Labo-ratory 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 0.2mg/day on Days 0, 2, 4, 6, and 8 AQ2 (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 / Cremo-phor EL / saline (2:1:7). The tumor size was meas-ured 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 sam-ples and mice organ tissues (including the lungs and kidneys) were resected, fixed with formalin and em-bedded 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 com-pound of high purity and yield. Its structure is shown in Figure1A. To test the biologic activity of 3-indole F1 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 concentra-tion (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
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Apoptotic Mechanism of 3-Indole Treatment/Lee et al 3
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 con-comitantly increased compared with the cells that were not treated with 3-indole (Fig. 2A). To further F2 dissect the apoptosis pathway induced by 3-indole we performed Western blot analysis for cytochrome-c release and caspase protein expression and used dif-ferent fluorogenic tetrapeptide substrates (Ac-DEVD-pNA, Ac-LETD-(Ac-DEVD-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 mito-chondria 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.
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.6,11,12ROSs are known to damage many mole-cules 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 mitochon-drial localization was detected by MitoTracker. In
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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 vari-ous 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 indi-cated times and cell proliferation was assessed by the MTT assay. Data shown are the means of 3 independent experiments; bars, SE. (C) 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 DMSO or 3-indole at the indicated con-centrations for 24 hours. (D) Cell apoptosis assay using electrophoresis indi-cated that an apoptotic DNA ladder appeared in various human lung cancer cells at 24 to 48 hours.
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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 produc-tion 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
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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 trea-ted with DMSO or 30 lM of 3-indole in medium for 0, 4, 8, and 12 hours. Blotting experiments were repeatrea-ted 3 times with similar results. (B) Induction of cas-pase activity by A549 cells. Cells were treated with 30 lM 3-indole for the indicated times and lysed in cascas-pase buffer. Enzymatic activities of cascas-pase-3, -8, and -9 proteases were determined with fluorogenic substrates using corresponding caspase colorimetric assay kits. Fold induction in caspase activity was cal-culated 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;
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 trea-ted with DMSO or 30 lM of 3-indole in medium for 0, 4, 8, and 12 hours. Blotting experiments were repeatrea-ted 3 times with similar results. (B) Induction of cas-pase activity by A549 cells. Cells were treated with 30 lM 3-indole for the indicated times and lysed in cascas-pase buffer. Enzymatic activities of cascas-pase-3, -8, and -9 proteases were determined with fluorogenic substrates using corresponding caspase colorimetric assay kits. Fold induction in caspase activity was cal-culated 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;