透過活化p53引起人類非小細胞肺癌與肝癌細胞凋亡的吲哚喹嚀基藥物與其作用機制的探討

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(1)國立臺灣師範大學生命科學系博士班研究生論文. 透過活化 p53 引起人類非小細胞肺癌與肝癌細胞凋亡的吲哚喹嚀基藥物與其作用機制的探討 Study of a novel indolylquinoline compound that induces apoptosis in human non-small cell lung cancer and hepatocellular carcinoma cells through p53 activation. 研究生 : 劉俊彥 Chun-Yen Liu. 指導教授 : 方剛博士 Kang Fang. 中華民國一○五年七月.

(2) LIST OF ABBREVIATIONS ADAMs ANOVA ATCC BCA DAPI DCFDA DMSO DMEM DSBs DTT ECL ECM EDTA EMMQ EMT FBS FITC HDAC HCC IC50 JC-1 MAPK MMP (ΔΨm) MMP MTT NS shRNA NSCLC PARP PBS PCNA PI3K PI ROS RNAi SCLC. A Disintegrin And Metalloproteinase Analysis of variance American Type Culture Collection Bicinchoninic acid 4',6-Diamidino-2-phenylindole 2`,7`-Dichlorofluorescein diacetate Dimethyl sulfoxide Dulbecco’s Modified Eagle Medium Double-strand DNA breaks Dithiothreitol Enhanced Chemiluminescence Extracellular matrix Ethylenediaminetetraacetic acid 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline Epithelial Mesenchymal Transition Fetal bovine serum Fluorescein isothiocyanate Histone deacetylases Hepatocellular carcinoma The half maximal inhibitory concentration 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl benzimidazolocarbocyanineiodide. Mitogen-Activated Protein Kinase Mitochondrial Membrane Potential Matrix Metalloproteinase 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide Non-specific shRNA Non-small-cell-lung-cancer Poly(ADP-ribose) polymerase Phosphate-buffered saline Proliferating cell nuclear antigen Phosphoinositide 3-kinase Propidium iodide Reactive oxygen species RNA interference Small cell lung cancers.

(3) SDS-PAGE shRNA TSG TRITC TEMED. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Short hairpin RNA Tumor suppressor genes Tetraethyl rhodamine isothiocyanate Tetramethylethylenediamine.

(4) Index Page Part Ⅰ I.. ABSTRACT ................................................................................ 5. II.. 中文摘要 ..................................................................................... 7. III.. INTRODUCTION ....................................................................... 8 1. Lung cancer ............................................................................................. 8 2. Classification of lung cancer.............................................................. 8 3. Small cell lung cancer .......................................................................... 8 4. Non-small cell lung cancer ................................................................. 8 5. Squamous cell (epidermoid) carcinoma ........................................ 8 6. Adenocarcinoma .................................................................................... 9 7. Large cell (undifferentiated) carcinoma ....................................... 9 8. Other subtypes........................................................................................ 9 9. Apoptosis and p53 ................................................................................. 9 10. Clinical treatment................................................................................ 11 11. Novel synthesis of indolylquinoline derivative .......................... 12 12. Aims of the study ................................................................................. 13. IV.. MATERIALS AND METHODS ............................................... 14 1. Chemicals and cell culture. .............................................................. 14 2. Cell viability assay. .............................................................................. 14 3. Colony forming assay. ........................................................................ 15 4. Comet assay. .......................................................................................... 15 5. Determination of apoptosis. ............................................................. 16 Double staining with Annexin V-FITC and PI. ........................ 16 Cell-cycle distribution........................................................................ 16 6. Determination of ΔΨm. ..................................................................... 17 7. Release of cytochrome c release. .................................................... 17 8. Western blot analysis. ........................................................................ 18 9. Transfection with p53 shRNA......................................................... 18 10. Xenograft tumor evaluation. ........................................................... 19 11. Cell migration assay. .......................................................................... 20 12. Gelatin zymography ........................................................................... 20 1.

(5) 13. Statistical analysis. .............................................................................. 21 V.. VI.. RESULTS. ................................................................................. 22 1. EMMQ inhibits cell proliferation in both A549 and H460 cell………………………………………………………………….22 2. EMMQ-induced DNA damage in wild-type p53 cell lines were examined by comet assay. ............................................................... 22 3. The EMMQ increased sub-G1 population cells, G2/M arrest and apoptosis in both A549 and H460 cells. ....................................... 23 4. EMMQ-induced apoptosis through intrinsic pathway. ......... 24 5. The suppressed growth of xenograft tumors in EMMQ-treated A549 cells. ....................................................................... 25 6. The extent of EMMQ-induced apoptosis is dependent on p53 status................................................................................................................. 26 7. Down-regulated p53 abolished the onset of EMMQ-induced cell death in NSCLC cells .......................................................................... 27 8. The effect of EMMQ inhibited metastasis is dependent on Akt and β-catenin status............................................................................ 28 DISCUSSION ............................................................................. 30. VII. CONCLUSION .......................................................................... 35 VIII. FIGURE AND LEGENDS .................................................................... 36 Figure 1 The structure of a new synthetic compound EMMQ and its inhibitory effect on cell growth of wild type p53 NSCLC cell lines. .......................................................................................................... 36 Figure 2 EMMQ treatment inhibits growth of wild type p53 NSCLC cell lines........................................................................................... 38 Figure 3 EMMQ treatment induces DNA damage in A549 and H460 cells. ....................................................................................................... 40 Figure 4 EMMQ treatment induces change in cell cycle distribution in A549 and H460 cells. ..................................................... 42 Figure 5 Apoptosis induced by EMMQ in NSCLC cell lines. .. 45 Figure 6 EMMQ treatment induces apoptotic cell death in intrinsic pathway related proteins in A549 and H460 cells........... 47. 2.

(6) Figure 7 EMMQ treatment causes apoptosis through reduced ΔΨm and enhanced cytochrome c release in A549 and H460 cells... ................................................................................................................ 48 Figure 8 EMMQ inhibits growth of xenograft tumors by A549 cells in nude mice. ........................................................................................ 50 Figure 9 EMMQ treatment causes apoptosis through reduced ΔΨm on H1299 cells with stable expression of ectopic p53 cells. 53 Figure 10 EMMQ treatment induces change in cell cycle distribution on H1299 cells with stable expression of ectopic p53…................................................................................................................. 55 Figure 11 EMMQ treatment causes apoptosis through enhanced cytochrome c release on H1299 cells with stable expression of ectopic p53. .......................................................................... 57 Figure 12 EMMQ treatment on p53 silencing in A549 and H460 cells proliferation. ........................................................................................ 59 Figure 13 EMMQ treatment reduces apoptotic cell death in intrinsic pathway related proteins on p53 silencing in A549 and H460 cells. ....................................................................................................... 61 Figure 14 EMMQ inhibits cell migration of A549, H1299 and H460 cells. ....................................................................................................... 62 Figure 15 Proposed mechanism for EMMQ-induced apoptotic cell death in NSCLS cells. ......................................................................... 65. 3.

(7) Part II I.. ABSTRACT ............................................................................................... 66. II.. 中文摘要 ................................................................................... 67. III.. INTRODUCTION ..................................................................... 68 1. Liver cancer ........................................................................................... 68 2. Aims of the study ................................................................................. 68. IV.. MATERIALS AND METHODS ............................................... 70 1. Cell culture and reagent.................................................................... 70 2. Cell growth assay. ................................................................................ 70 3. Colony forming assay. ........................................................................ 70 4. Comet assay. .......................................................................................... 71 5. Determination of apoptosis. ............................................................. 71 Double staining with Annexin V-FITC and PI. ........................ 71 Cell-cycle distribution........................................................................ 71 6. Measurement of intracellular ROS............................................... 72 7. ΔΨm assay. ............................................................................................. 73 8. Analysis of cytochrome c release. .................................................. 73 9. Western blot analysis. ........................................................................ 74 10. Transfection with p53 shRNA......................................................... 74 11. Isolation of mitochondria and cytosol fractions. ...................... 75 12. Statistical analysis. .............................................................................. 75. V.. RESULTS. ................................................................................. 76 1. EMMQ inhibited cell proliferation in human liver cancer cells…. .............................................................................................................. 76 2. The EMMQ increased sub-G1 population cells, G1 arrest and apoptosis in HepG2 cells. .......................................................................... 76 3. EMMQ induces HepG2 cell apoptosis by way of DNA damage .. ......................................................................................................... 78 4. EMMQ-induced HepG2 cell apoptosis through intrinsic pathway of ΔΨm drop and cytochrome c release ............................. 79 5. EMMQ induces HepG2 cell apoptosis through ROS production. ..................................................................................................... 80 6. EMMQ-induced apoptosis through intrinsic pathway. ......... 81 4.

(8) 7. Down-regulated p53 abolished the onset of EMMQ-induced cell death in hepatocellular carcinoma cells ....................................... 81 VI.. DISCUSSION ............................................................................. 83. VII. CONCLUSION .......................................................................... 87 VIII. FIGURE AND LEGENDS .................................................................... 88. Figure 16 Effect of EMMQ on cell growth in HCC cell lines and human hepatic cells. .................................................................................... 88 Figure 17 EMMQ treatment induces change in cell cycle distribution, G1 phase cell cycle arrest and apoptosis ratio on HepG2 cells..................................................................................................... 91 Figure 18 EMMQ treatment induces DNA damage in HepG2 cell lines . ......................................................................................................... 96 Figure 19 EMMQ induces ROS causes apoptosis through reduced ΔΨm and enhanced cytochrome c release in HepG2 cells…. .............................................................................................................. 99 Figure 20 EMMQ treatment induces apoptotic cell death in intrinsic pathway related proteins in HepG2 cells. ........................ 102 Figure 21 Effects of EMMQ treatment by p53 silencing in HepG2 cells................................................................................................... 104 Figure 22 EMMQ treatment reduced DNA damage by p53 silencing in HepG2 cells ........................................................................... 107 Figure 23 Proposed a model for EMMQ mediated apoptosis in HepG2 cells. ................................................................................................. 109 IX.. REFERENCES ........................................................................ 110. 5.

(9) Part Ⅰ I. ABSTRACT Lung cancer is the leading cause of cancer mortality worldwide. NSCLC accounts for about 75% to 80% of lung cancer cases. Chemotherapy adverse side effects and resistance to current anticancer agents have been the pressing problems in the success of lung cancer therapy. Anticancer drugs are in urgent need especially for lung cancer. A program to develop a new anti-lung cancer agent by screening novel synthetic compounds was initiated. EMMQ was selected by MTT cell viability assay. The cell growth rate of A549 and H460 NSCLC cells was reduced by a low concentration of EMMQ treatment. Our study revealed that EMMQ induced apoptosis in NSCLC cells with wild-type p53, while the drug is less potent to against p53-null cells. The study elucidated that EMMQ-induced apoptosis is was mediated through the intrinsic pathway involving DNA damage, activation of p53, interference of mitochondrial ΔΨm that led to release of cytochrome c and down regulation Bcl-2, activation of caspase family proteins, and finally cleavage of PARP polymerase cleavage. In vivo study showed that EMMQ reduced tumorigenesis and significantly suppressed growth rate of xenograft tumors in nude mice. In addition, metastasis studies demonstrated that EMMQ may inhibit wild-type p53 cells migration at low concentration. In conclusion, EMMQ was demonstrated as an effective p53 regulator in NSCLC cells. Our findings indicate that EMMQ may serve as a promising new and potential therapeutic agent for human lung cancers.. Keywords: Lung cancer, p53, apoptosis. 6.

(10) II. 中文摘要肺癌是全世界癌症中死亡率第一的癌症。非小型細胞肺癌（NSCLC）佔肺癌病患的比例約 75％至 80％。化療所造成的副作用與抗藥性在肺癌治療中是有待處理的棘手問題。因此發展出新的抗癌藥物對肺癌的病患是有必要的。此篇論文旨在篩選可抑制肺癌細胞生長新穎的合成化合物。透過 MTT 的分析方式鑑定 EMMQ 對非小型肺癌細胞的 IC50 的濃度。數據顯示低濃度的 EMMQ 即可減少 A549 和 H460 此兩種非小型肺癌細胞的生長速率。研究也證明，EMMQ 可誘導具有正常 p53 基因的非小細胞肺癌的細胞凋亡，而且該藥物對 p53-null 的非小細胞肺癌則無明顯的效果。研究顯示 EMMQ 會誘導細胞 DNA 損傷，活化 p53 蛋白，干擾粒線體的膜電位而釋放細胞色素 c，造成 Bcl-2 的下降，活化 caspase-3，讓 PARP 裂解而造成的內生性細胞凋亡。活體的實驗證明，EMMQ 可以抑制裸鼠異種移植腫瘤的生長。最後，此研究證實 EMMQ 可在低濃度時活化非小細胞肺癌細胞的 p53 而造成細胞凋亡。此外，本研究還發現 EMMQ 可抑制具有正常 p53 基因之非小細胞肺癌細胞的細胞轉移。因此本論文的數據顯示 EMMQ 可能成為一種新穎且有潛力的人類肺癌治療藥物。關鍵字: 肺癌, p53, EMMQ, 細胞凋亡. 7.

(11) III. INTRODUCTION 1. Lung cancer Lung cancer has been the most common cancer for several decades 1.61 million new cases were forecast that represented 12.7% of all new cancers in the world [1]. It is the leading cause of cancer death in the world. Lung cancer is the second most common cancer in both men and women in 2014 in the United States [2, 3]. In Asia, more than 30% of patients with lung cancer are non-smokers and about 50% of lung cancers occur in women [4, 5].. 2. Classification of lung cancer Lung cancers are able to start in the cells lining the bronchi and parts of the lung such as the bronchioles or alveoli. The first changes in many genes inside the lung cells cause the cells to grow faster [6]. Lung cancer has been classified as SCLC and NSCLC [6-8].. 3. Small cell lung cancer About 10% to 15% of all lung cancers are SCLC, named for the size of the cancer cells. SCLC often begins in the bronchi near the center of the chest, and it tends to spread widely through the body fairly early in the course of the disease [9, 10].. 4. Non-small cell lung cancer There are several subtypes of NSCLC, predominantly adenocarcinoma, squamous-cell carcinoma, and large-cell carcinoma. 8.

(12) SCLC represents roughly 15% of all pulmonary cancers.. 5. Squamous cell (epidermoid) carcinoma: Approximately 25% to 30% of all lung cancers are squamous cell carcinomas. These cancers start in early versions of squamous cells, which are flat cells that line the inside of the airways in the lungs. They are often linked to a history of smoking and tend to be found in the middle of the lungs, near a bronchus [8, 10].. 6. Adenocarcinoma: About 40% of lung cancers are adenocarcinomas. This type of lung cancer is the most common type of lung cancer appearing in non-smokers. [8, 10].. 7. Large cell (undifferentiated) carcinoma: This type of cancer can appear in any part of the lung. It tends to grow and spread quickly, hence making it harder to treat [8].. 8. Other subtypes: There are also a few other subtypes of NSCLC, such as adenosquamous carcinoma and sarcomatoid carcinoma [8, 10].. 9. Apoptosis and p53 Apoptosis is a regulated cellular death mechanism by nuclear condensation, cell shrinkage, membrane blabbing, and DNA damage [11]. 9.

(13) The apoptotic pathway occurs normally during development, aging and in the maintenance of tissues homeostatic mechanism. It also occurs as a defense mechanism such as immune reactions or when cells are damaged by disease or poisonous agents [12]. Apoptosis is triggered by a wide variety of signals. Three main types of biochemical changes are observed in apoptosis: activation of caspases, DNA and protein breakdown and membrane changes and recognition by phagocytic cells [13]. Apoptosis is also triggered by internal signals and external signals. The execution phase of apoptosis involves the activation of caspases. The upstream caspase for the intrinsic pathway is caspase-9 and extrinsic pathway is caspase-8. Cell cycle is one way that may be achieved through the coupling of the cell cycle and programmed cell death [14]. In fact, defects in apoptotic pathways are now thought to contribute to a number of human diseases, ranging from neurodegenerative disorders to malignancy [15]. Cancer is one of the scenarios where cells are more resistant to apoptosis, resulting in malignant cells that will not die. Therefore, apoptosis plays an important role in the treatment of cancer as it is a popular target of many treatment strategies [16].. The p53 gene is the first TSG linked to apoptosis. P53 mutations occur in the majority of human tumors and are often associated with advanced tumor stage and poor patient prognosis. The p53 is one of TSG and located at chromosome 17p13.1. It codes a 53 kDa nuclear protein and acts as a DNA-binding, sequence-specific transcription factor and activates the expression of genes engaged in promoting growth arrest in 10.

(14) the G1 phase or cell death in response to the genotoxic stress [17-19]. TSG p53 blocks cells at the G2 checkpoint and inhibits activity of cdc2, a cyclin-dependent kinase is required to enter mitosis [20, 21]. The function of p53 to inhibit cellular proliferation or to induce apoptosis is suppressed by HDM2, the human homologue of the MDM2 [22, 23]. MDM2 blocks p53 regulation of target genes and enhances its proteasome dependent degradation [23, 24]. Missense mutations (mainly G→T transversions) are clustered in the middle of the gene at codons 157, 245, 248, and 273 at p53. This change abolishes its tumor suppressing activity and extends the p53 mutant protein half-life that can be easily detected by immunohistochemistry [24-27]. In response to DNA damage or other types of stress, p53 acts as a sequence-specific transcription factor and orchestrates the appropriate cellular response by inducing cell-cycle arrest, apoptosis, senescence, or differentiation [28, 29].. 10. Clinical treatment Depending on the stage of the disease and other factors, the main treatment options for people with NSCLC include: surgery, radiofrequency ablation, radiation therapy, chemotherapy, targeted therapies and immunotherapy [30]. Anti-cancer chemotherapy treatment were done by injecting drug into a vein or taken by mouth. The drugs enter the bloodstream and go throughout the body, making this treatment useful for cancer anywhere in the body [30, 31].. The chemotherapy drugs most often used for NSCLC include: 11.

(15) Cisplatin, Carboplatin, camptothecin analogs, Paclitaxel (Taxol), Albumin-bound paclitaxel (nab-paclitaxel, Abraxane), Docetaxel (Taxotere), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Irinotecan (Camptosar), Etoposide (VP-16), Vinblastine and Pemetrexed (Alimta) [32, 33]. The camptothecin analogs, blocks DNA supercoils by inhibiting DNA topoisomerase I [34].. Gemcitabine, a nucleoside analog, represents one of the building blocks of nucleic acids [35]. Platinum agents induce DNA cross-linking and apoptotic cell death through binding with DNA. The cisplatin is currently used in lung cancer patients The drug was linked with DNA and inhibited cell growth, thus lead to cell death. In addition there are target therapies for gene mutation patients [36]. Iressa (Gefitinib) is the first selective inhibitor of EGFR tyrosine kinase domain. Taxol (Paclitaxel) is approved in the UK for treating ovarian, breast and lung, bladder, prostate, melanoma, esophageal, and other types of solid tumor cancers as well as Kaposi's sarcoma [37, 38]. Paclitaxel induces cell type-dependent p53, p21 and G1/G2 arrest, thus inhibits mitosis and induces apoptosis [39-41]. In this dissertation, we focused on the role of p53 and its relationship with cancer therapy agent EMMQ.. 11. Novel indolylquinoline derivative The indolylquinoline derivative contains both indole and quinoline subunits. Anti-leishmanial activities were found with indolylquinoline derivatives. Leishmaniasis is a disease complex caused by 17 different 12.

(16) species of protozoan parasites belonging to the genus Leishmania [42]. Pentamidine and amphotericin B have been used as treatment of leishmaniasis for many years. Recently reports demonstrated the antileishmanial activity of the indolylquinoline derivatives both in vitro and in vivo [43].. 12. Aims of the study The study is to determine if EMMQ is a potential alternative to treat human lung cancer cells by examining the growth rate of cells and xenograft tumors.. 13.

(17) IV. MATERIALS AND METHODS 1. Chemicals and cell culture The synthetic compound, EMMQ MW 298.4, the structure shown in Fig 1A) [44], was purified to more than 98% purity and dissolved in DMSO at 10 mM for storage. Three cell lines, H460 (HTB177TM), H1299 (CRL5803TM) and A549 (CCL185TM) of human NSCLC cells and MRC-5 of human Lung fibroblast cells (CCL-171 TM) were acquired from ATCC and maintained in DMEM and cultured with L-glutamine, sodium pyruvate, and supplied with 10% heat-inactivated FBS under 5% CO2 at 37 °C. The selected stable H1299 clones transfected with cytomegalovirus promoter-driven pcDNA-p53 encoding full-length wild type p53 (H1299/p53) or mutant p53R267P (H1299/p53R267P) were maintained in 10 % serum-supplemented DMEM. Short hairpin RNA targeting p53 and scrambled non-specificity was acquired from the National RNAi Platform, Academia Sinica, Taipei, Taiwan.. 2. Cell viability assay The cell cytotoxicity was measured as previously described [45] . Briefly, cells were cultured at 1.5×103 cells per well in 96-well plates. The attached cells supplemented with minimal amounts of FBS were treated with different concentration of cisplatin (Sigma), EMMQ or vehicle control DMSO at 37°C for 48 h. Cells were then kept in 10 μL of MTT (5 mg/mL) or cisplatin with medium at 37 °C for 4 h. MTT was then removed, added with 100 μL DMSO into each well and the absorption measured by measuring the absorbance at 570 nm wavelength 14.

(18) with a 96-well microplate reader (Thermo Fisher Scientific, USA).. 3. Colony forming assay Cells were seeded at 50 cells per well in a 6-well plates for 16 h to allow attachment and then added with EMMQ at different concentrations or vehicle control for 48 h. After replacing with fresh media and growing in 3 mL medium for 14 days, the colonies were fixed before staining with 0.5% methylene blue in ethanol for 4 h. The size and number of stained colonies with more than 50 cells were counted under inverted phase contrast microscope. Colony formation was calculated as a percentage to untreated control cultures.. 4. Comet assay Conventional slides were covered with a layer of 70 μL 0.5 % normal agarose and 0.5% low melting point agarose (GIBCO-BRL). A volume of 70 μL of low melting point agarose (0.5%, w/v) (GIBCO-BRL) was mixed with approximately 2 × 104 cells suspended in 15 μL of media. The mixture was then layered onto the slides, and immediately overlaid with coverslips. After agarose solidification at 25 °C for 30 min, the coverslips were removed and the slides immersed 60 min at 4 °C in fresh lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10 and 1% Triton X-100). The slides were soaked in the alkaline buffer (300 mM NaOH and 1 mM Na2EDTA at pH 13) on the ice bath for 20 min and electrophoresed (30 V and 300 mA) for 20 min. Slides were then transferred to the neutralization buffer (0.4 M Tris-HCl at pH 7.5) on ice 15.

(19) bath for 15 min. Finally, slides were dried in methanol for 5 min and stained with 50 μL of PI (4 µg/mL). The tails were observed under a fluorescence microscope (Nikon, Japan) and quantified by using CometScore™ software (Tritek Corp, Sumerduck, VA).. 5. Determination of apoptosis Double staining with Annexin V-FITC and PI Cells were seeded at 2 ×105 cells per well in 12-well plates and allowed to attach overnight. Cells were treated by various concentrations of EMMQ incubated at 37 °C for 48 h. DMSO containing medium served as the vehicle control. The cells were trypsinized and stained with1 μL annexin-V/FITC (20 µg/mL, BD Bioscience) and 1 μL of PI (50 µg/mL) at room temperature for 30 min in the dark. The early and late phase of apoptosis was measured by annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience). The flow cytometer FACS CaliburTM (BD Bioscience) was used for analysis and the data were analyzed using the FlowJo software (Tree Star, Inc.).. Cell-cycle distribution Cell cycle distribution was determined by suspending cells in 70 % ethanol and kept at -20 °C for 24 h prior to addition of 10 µg/mL of PI (Sigma, St. Louis, MO) and 10 µg/mL of RNaseA (ICN Pharmaceutical; Costa Mesa, CA) in PBS (UniRegion Bio-tech, Taiwan) for 30 min. The data as acquired by flow cytometry were analyzed with software FlowJo (Tree Star, Inc.). 16.

(20) 6. Determination of ΔΨm ΔΨm was determined using MitoPT™ JC-1 Assay Kit (ImmunoChemistry Technologies, Bloomington, USA). Briefly, the seeded cells were cultured in 2% serum-supplemented DMEM containing different concentration of EMMQ or vehicle control and incubated at 37 °C for the time points as indicated. The collected cells were washed with 1× assay buffer. After centrifugation at 1,000 rpm for 5 min, cell pellets were stained with 250 μL mixture containing 5 μL of JC-1 with 995 μL 1× assay buffer for 25 min at 37 °C. The residual JC-1 was removed by centrifugation at 1,000 rpm for 5 min and the pellet mixed with 1× assay buffer. JC-1 fluorescence was measured to assess the emission shift from green (530 nm) to red (590 nm) using 488 nm excitation wavelength. Data were given as the relative ratio of green to red fluorescence intensities, indicating the level of depolarization of the mitochondrial membrane potential. The data as determined by FACS CaliburTM were quantified and the expressed as the percentage of mitochondrial membrane potential drop relative to those of untreated cells.. 7. Release of cytochrome c release The harvested cells after treatment were treated with 100 μL digitonin (50 µg/mL PBS, 100 mM potassium iodide and 1 mM EDTA) for 5 min on ice until more than 95 % of cells permeabilized. Cells were then fixed and stained with 3.7 % formaldehyde and DAPI (1:3,000) (Sigma, USA) in PBS for 20 min at room temperature, washed thrice in 17.

(21) PBS, and incubated in blocking buffer (3% bovine serum albumin (Themo, USA) and 0.05 % saponin in PBS) for 1 h. The cells were incubated overnight at 4°C with anti-cytochrome c mouse monoclonal antibody (BD PharMingen) that was diluted to 1:200 in blocking buffer, washed thrice, and incubated for 1 h at room temperature with TRITC-conjugated goat anti-mouse (Santa Cruz) in blocking buffer. The cells were then counterstained with Mitotracker Green (Invitrogen Life Technologies). The samples were detected using a Leica TCS SP5 Confocal Spectral Microscope.. 8. Western blot analysis The western blot analysis was determined by electrophoresis of the protein contents of cell lysates were collected and the concentrations quantitated using BCA assay (Pierce Biotechnology, Rockford, IL). A total of 20 µg of protein were resolved by electrophoresis through SDS-PAGE gel was transferred to nitrocellulose filters, blocked with 5% of Skim Milk (BD, Mansfield, MA) and incubated with primary and secondary antibodies. The emitted chemiluminescence signals were visualized by ECL detection kit (Millipore).. 9. Transfection with p53 shRNA A549 and H460 NSCLC cancer cells were seeded in 60-mm dishes at 5 × 105 cells/dish, incubated overnight, and then transfected with p53 shRNA using Lipofectamine 3000 transfection reagent (Invitrogen, USA) according to the manufacturer’s protocol. After a 24 h transfection period, 18.

(22) cells were treated with EMMQ for 48 h. Cell lysates were collected for western blot analysis.. 10. Xenograft tumor evaluation The athymic nu/nu female mice (BALB/c) of 3-4 week of age were obtained from the National Laboratory Animal Center (Tainan, Taiwan) and housed under pathogen-free conditions with a 12 h light/12 h dark schedule in the Animal Resource Facility at the animal center (Kaohsiung Medical University, Kaohsiung, Taiwan) in accordance with the Institutional Animal Care and Use Committee guidelines. The animal study was performed according to protocols of the Institutional Animal Guidance. A total of 1×106 cells were suspended in 100 μL of PBS and were injected into dorsal legs hypodermic area of nude mice with a total of 6 mice in each group. The EMMQ was dissolved in DMSO and the mixture of PBS and Matrigel™ Basement Membrane Matrix (BD Biosciences, San Jose, CA) (4:1) before EMMQ treatment. Once the tumors reached 50 mm3, the mice were injected subcutaneous with 200 μL of EMMQ (at a dosage of 1 mg/kg/mouse) or vehicle control twice a week for four consecutive weeks. The size of each tumor was measured at each time point before EMMQ administration. The tumor volumes were calculated according to the formula: 1/2 × (length×width2). After 35 days of drug treatment, mice were sacrificed by CO2 inhalation and death was confirmed by cervical dislocation. The tumor samples were dissected were measured and lysed later for protein analysis. One-way ANOVA test was used for statistical comparisons between different groups. 19.

(23) 11. Cell migration assay Migration was determined using the wound healing assays by A549, H1299 and H460 cells, respectively. Cells were seeded at 2 ×105 cells per well into 12-well plates for 16 h to allow cell attachment. Each NSCLC cells were treated different concentrations of EMMQ or solvent control DMSO and scraped with a 200 µL tip (time 0). Before imaging, suspended cells were washed off. The distance of migrating cells was measured from images at 48 h after EMMQ treatment. The results of wound healing assay were normalized as ratio of wound repaired area to the non-treated control set to 100%.Wound closure was evaluated and photographed at 48 h with an inverted microscope (Nikon, Japan).. 12. Gelatin zymography A549 cells were starved for 24 h with serum-free medium. Subsequently, cells in media containing 0.5% FBS were treated with EMMQ for different time periods and concentrations, and thereafter, the supernatants were collected. The samples were analyzed with gelatin zymography (0.1% w/v) to assess the enzymatic activity of MMPs by using gelatin as the substrate. Each lane was loaded with a total protein concentration of 3 µg and subjected to SDS-PAGE electrophoresis (30% acrylamide, 10% SDS, 10% APS, TEMED, 1.5 M Tris (pH 8.8), 1.0 M Tris (pH 6.8)) at 48 °C. Gels were washed twice in 50 mM Tris (pH 7.4) containing 2.5% (v/v) Triton X-100 for 1 h, followed by repeated 10-min rinses in 50 mM Tris (pH 7.4). Gels were then incubated overnight in 50 20.

(24) mM Tris (pH 7.5) containing 10 mM CaCl2, 0.15 M NaCl, 0.1% (v/v) Triton X-100, and 1% NaN3 at 37 °C with gentle shaking overnight. After incubation, gels were stained with 0.25% Coomassie brilliant blue and destained in 7.5% acetic acid with 20% methanol. Matrix metalloproteinases in the loaded supernatants leads to the gelatinase bands appearing white on a blue background.. 13. Statistical analysis Experiments were performed 3 times. The differences between the treated and control cells were analyzed using the Student's t-test between two groups. The data were expressed as mean values ± SD of three independent experiments and p<0.05 was considered significant.. 21.

(25) V. RESULTS 1. EMMQ inhibits cell proliferation in both A549 and H460 cells To investigate the cytotoxic effect of EMMQ and to compare it with that of cisplatin in lung cancer cells, MTT assays were performed after treatment of NSCLC cells with EMMQ or cisplatin. Cisplatin reduced H460 cell growth in dose-dependent manners and cell death over 50% at 50 µM (Fig. 1B). Approximately over 100 synthetic compounds for the lung cancer screen with a known molecular weight were carried out by MTT assay. EMMQ was selected for the highest cytotoxic effect on the NSCLS cell lines. However, EMMQ induced cell death by more than 50% at 10 µM in A549 and H460 cells. The IC50 values of A549 and H460 cells were reduced with 9.7 and 9.5 μM, respectively; while no apparent growth inhibition shown in MRC-5 and H1299 cells within the concentrations studied (Fig. 1C). The results suggested that both wild type p53 NSCLC cell lines were more sensitive to EMMQ than to cisplatin.. The colony formation capacity is significantly decreased in both A549 and H460 cells by EMMQ treatment (Fig. 2A and 2B). The numbers of colonies with in A549 and H460 cells were reduced by more than 50% when treated with 8 μM EMMQ, respectively, however there seemed no obvious inhibitory effect on null p53 H1299 cells (Fig. 2B). 2. EMMQ-induced DNA damage in wild-type p53 cell lines were examined by comet assay 22.

(26) To determine whether EMMQ induced DNA damage in NSCLC cells in vitro using comet assay. The results showed that EMMQ induced DNA damage in A549 and H460 cells with wild-type p53 status. At 5 μM concentrations of EMMQ led to A549 cells a long DNA migration smear (comet tail), and these effects occurred in a dose-dependent manner (Fig. 3A), but the similar effect on H460 cells occurred at the highest dosage (10 μM). The comet tails indicating DNA lesions in A549 and H460 cells were detected after 24 h EMMQ treatment and the appearance of the excluded tail length was dose-dependent (Fig. 3B).. 3. EMMQ increased sub-G1 population cells, G2/M arrest and apoptosis in both A549 and H460 cells To gain more knowledge on cell cycle distribution following treatment, EMMQ induced cell cycle disturbance in NSCLC cells were examined. After 48 h EMMQ treatment, the cells were stained by PI and the cell cycle distribution was detected by flow cytometry. The sub-G1 cell population significantly increased in A549 and H460 cells than in DMSO vehicle controls and the effects were dose-dependent, while no effect shown in H1299 cells (Fig. 4A). In addition, our data showed that EMMQ induced A549 and H460 cells cell cycle arrest in G2/M in 8 μM at 24 h. The results indicated that EMMQ induced cell cycle arrest in G2/M at 24 h and later apoptosis at 48 h in wild type p53 A549 and H460 cells (Fig. 4B).. To examine whether EMMQ treatment induces apoptosis, NSCLC 23.

(27) cells are treated with various concentrations of EMMQ (0, 5, 8, and 10 µM) and then conducted the flow cytometry-based Annexin V and PI double staining assay. Starting at a concentration of 5 μM after 48 h treatment of EMMQ, the early and late apoptotic phase cell population rose to 13% and 16% for A549 cells as well as 6% and 5% for H460 cells, respectively. However, no apoptotic cell death was shown in null p53 H1299 cells (Fig. 5A and 5B). The results implied that EMMQ-induced cell viability reduction in NSCLC cells carrying wild-type p53 was caused by apoptotic cell death following DNA damage.. 4. EMMQ-induced apoptosis through intrinsic pathway The apoptosis attributed to DNA damage can proceed through intrinsic pathway or extrinsic pathway [12, 29]. To clarify whether the intrinsic pathway is involved in EMMQ-induced apoptosis, we firstly examined the protein expression of p53, anti-apoptotic Bcl-2 family protein (Bcl-2), caspase-3 and cleavage of poly(ADP ribose) polymerase (PARP) by western bolt. Fig. 6A shows the increased concentrations of EMMQ activated p53 and down-stream effector p21, reduced p-AktS473, Bcl-2 and procaspase-3 levels, and increased cleavage of poly(ADP ribose) polymerase (PARP) in A549 and H460 cells after 48 h treatment. On the other hand, in the presence of 5 μM of EMMQ, activation of p53, reduction of p-AktS473 and Bcl-2 intensities as well as procaspase-3 dissipation and increased PARP cleavage in A549 and H460 cells were detected in time-dependent manners. No change was shown in Akt and p-AktS473, Bcl-2 levels and procaspase-3 as well as cleavage PARP in 24.

(28) p53-null H1299 cells within the time intervals and drug concentration ranges as studied (Fig. 6B). The results of western blots implied that EMMQ induced apoptosis through p53 activation and cleavage of PARP and diminished procaspase-3 is related to intrinsic pathway.. Mitochondria-related apoptotic pathway was linked to signal cascade following the ΔΨm disruption, signaling mitochondrial dysfunction involvement [46]. It’s well known that the attenuated ΔΨm, outer membrane regulator Bcl-2 and release of downstream modulator mitochondrial cytochrome c indicated mitochondria-mediated pathway leading to apoptosis [47, 48]. Fig. 7A shows ΔΨm loss in A549 cells suggested that the mitochondria-mediated apoptosis by low dosage of EMMQ was initiated starting at 4 h, but the effect was absent in H1299 cells. The impaired mitochondrial functions were further accentuated by cytochrome c release in both A549 and H460 cells with increasing drug concentrations after 24 h treatment (Fig. 7B).. 5. The suppressed growth of xenograft tumors in EMMQ-treated A549 cells To assess the effect of EMMQ in vivo, a tumor xenograft study (6 animals per group) was carried out. Subcutaneous injection of EMMQ twice a week at a concentration of 1 mg/kg per mouse resulted in a significant decrease in tumor volumes. There was also a notable decrease in the size and excised volume of tumors from EMMQ-treated mice as compared with control (Fig. 8A, 8C and 8D). The size and the excised 25.

(29) volume of tumors decreased to more than 50% following 4 week treatment, while no significant difference of body weight in EMMQ-treated nude mice relative to vehicle control as shown in Fig. 8B. The lysates from the dissected tumors were further analyzed by western blot analysis. The levels of survival genes Akt, p-AktS473 and cell proliferation marker PCNA as well as mitochondrial modulator Bcl-2 were noticeably reduced. The intensities of p53 and cleaved PARP were elevated, whereas those of procaspase-3 decreased in EMMQ-treated group as compared with those of control treatment (Fig. 8E).. 6. The extent of EMMQ-induced apoptosis is dependent on p53 status To examine the role of p53 played during the process of EMMQ induced apoptosis. H1299 cells with stable expression of ectopic p53 (H1299/p53) (positive control) or mutant p53R267P (H1299/p53R267P) (negative control) were established [49]. EMMQ induced cell viabilities significantly decrease in H1299/p53 cells as compared with H1299/p53R267P clone (Fig. 9A). Fig. 9B shows the loss of ΔΨm in H1299/p53 cells suggested that the mitochondria-mediated apoptosis occurred in low dosage of EMMQ, but the effect was absent in H1299/p53R267P clone.. Furthermore, the induced apoptotic sub-G1 cell populations by EMMQ were more apparent in H1299/p53 cells (Fig. 10). A substantial amount of cells remained at G2/M phase in H1299/p53R267P clone. The apoptotic cells induced by EMMQ were more distinct in H1299/p53 26.

(30) than those of H1299/p53R267P clone as determined by annexin-V/PI-staining (Fig. 11A). The release of cytochrome c by EMMQ in H1299/p53R267P was comparably less than that of H1299/p53 cells (Fig. 11B).. The apoptotic cells induced by EMMQ were more distinct in H1299/p53 than those of H1299/p53R267P clone as determined by annexin-V/PI-staining (Fig. 11A). The release of cytochrome c by EMMQ in H1299/p53R267P was comparably less than that of H1299/p53 cells (Fig. 11B). The results demonstrated that no significant effects of sub-G1 population, apoptosis ration and cytochrome c release level were detected during EMMQ concentration (0, 5, 8 and 10 μM) in H1299/p53R267P cells (Fig. 9, 10 and 11).. 7. Down-regulated p53 abolished the onset of EMMQ-induced cell death in NSCLC cells To ensure that p53 was indeed necessary in drug-mediated cell death, experiments by transfecting shRNA targeting exon 7 of p53 to cells prior to drug treatment were carried out along with those of NS control. The result of cell viability indicated that A549 and H460 cells were transfected with p53 shRNA led to the sensitivity toward EMMQ was eliminated as compared with cells transfected with NS control (Fig. 13).. Western blot analysis of A549 cells showed that cells introduced with p53 shRNA exhibited significant reduction of p53 as compared with 27.

(31) those transfected with NS control alone. In addition, the mitochondria modulator Bcl-2 and pro-survival gene p-AktS473 were unaffected by EMMQ by knocking down p53 (Fig. 14A). Similar results were also observed with reduced viabilities and Bcl-2 attenuation (Fig. 14B) in p53 shRNA-transfected H460. The results altogether suggested that p53 was needed during mitochondrial pathway activation that predates the effectiveness of EMMQ in motivating apoptotic cell death of NSCLC cells.. 8. The effect of EMMQ inhibited metastasis is dependent on Akt and β-catenin status To gain more knowledge on cell metastasis following treatment, we examined whether EMMQ inhibited NSCLC cell migration. The extensive evidence suggests that Akt-transduced signals directly influence cell motility in normal development and in disease [50]. These evidences demonstrated that the Akt inhibited cell motility and metastasis in cancer cells [51, 52]. In addition, β-catenin regulates the expression of genes involved in cell migration and adhesion, such as cadherins, catenins, ADAMs, and integrins, and this gene expression regulation is likely the mechanism by which β-catenin regulates cell motility and adhesion [53]. The results by western blot analysis demonstrated that EMMQ reduced the levels of Akt, p-AktS473 and β-catenin in A549 and H460 cells, but no change of protein expression level was detected in H1299 cells (Fig. 15A).. 28.

(32) The wound healing assay is measure cell migration in vitro. The basic steps involve creating a ‘‘scratch’’ in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells [54] . Fig. 14B showed that low dosage of EMMQ reduced A549 cells wound closure, however, the H1299 cells migrated into the wound and filled with the area at EMMQ treatment for 48 h. The result suggested that EMMQ may inhibit A549 cells migration at 5 µM.. MMPs are a group of proteolytic enzymes which are important in many physiological processes such as embryogenesis, development, and wound healing. Dysregulated MMP activity has long been implicated in diseases associated with uncontrolled proteolysis of connective tissue matrices such as arthritis, tumourgenesis and tissue ulceration. The results indicated that EMMQ may inhibit A549 and H460 cell migration at 5 µM. In addition, EMMQ reduced MMP-2 and MMP-9 activities of A549 cells in a dose and time manner (Fig. 14D). This implied that EMMQ may inhibit cells metastasis or possibly invasion in NSCLC.. 29.

(33) VI. DISCUSSION Chemotherapy adverse side effects and resistance to current anticancer agents have been the pressing problems in the success of lung cancer therapy [55]. As in the case of other chemotherapeutic agents, cisplatin therapy usually results in the second option, leading to programmed cell death, also known as apoptosis. Cisplatin is the most widely used anticancer drug and causes cell death by inducing apoptosis. Nevertheless, the high rate of resistance observed during therapy with cisplatin, as well as the occurrence of non-sensitive cancer cells, prompt the quest for agents endowed with apoptosis-independent mechanisms of action [56] . New chemotherapy strategies are urgently needed for lung cancer treatment.. Previous study showed that the IC50 value of a quinoline derivative, 6-methoxy-8-[(2-furanylmethyl)amino]-4-methyl-5-(3-trifluoromethylphe nyloxy)quinolone, is 16 ± 3 nM in breast cancer cells [57]. Another novel derivative of N-amidino-substituted benzimidazo[1,2-α]quinoline produced differential antiproliferative mechanisms in two human colorectal cancer cell lines that differ in p53 gene status [58]. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphen yloxy)quinoline induced apoptosis in T47D breast cancer cells through activation of caspase-8 and caspase-9 [59]. PQ15, 6-methoxy-4-methyl-8-[(4-quinolinylmethyl)amino]-5-(3-trifluoromethyl phenyloxy)-quinoline affected the survival pathway of T47D breast cancer cells [60]. 30.

(34) Among the indole derivatives, indole-3-carbinol mainly exists in cruciferous vegetables such as broccoli, cabbage, cauliflower, brussels sprouts, collard greens and kale was reported with inhibitory effects on prostate tumors through inhibiting proliferation, cell cycle progression and cell survival, thus induced cell death [61]. The combined indole-3-carbinol and genistein induced apoptosis in human colon cancer HT-29 cells [62]. Therefore, the possibility of combining indole with quinoline subunits with synergistic anticancer effect is of interest.. The reports showed that IC50 values of cisplatin for the A549 and H460 cells were 30 and 16 μM, respectively[63, 64]. Both Fig. 1B and 1C showed that IC50 values of cisplatin and EMMQ were 50 and 9 μM, respectively. In addition, EMMQ reduced cell viability by more than 50% at 9μM in A549 cells (Fig. 1C). Our data showed that, compared to cisplatin, EMMQ is more potent in inhibiting the growth of A549 and H460 cells. In many types of cancer, DNA repair systems have been entirely or partially switched off but not in the normal tissue cells [65]. Gene regulation concerning translation, transcription, cell cycle, and the functions were regulates in MRC-5 cells by UVB irradiation [66]. The study further validated that DNA repair system is maintained.in MRC-5 and H1299 cells, but not in A549 and H460 cells as indicated by comet assay (Fig 3A and 3B). The deficient DNA integrity accounts for the sensitivity and the effectiveness of EMMQ.. 31.

(35) The results showed that the sensitivity of the EMMQ in A549 and H460 cells is dependent on status of p53 that led to reduction of cell viability and tumor growth through apoptotic cell death (Fig. 1B, 8A and 9B). The EMMQ induced apoptosis in A549 and H460 cells at low concentrations was attributed to transient p53 elevation and PARP cleavage because of mitochondria perturbation (Fig. 6 and 7). Put altogether, the results suggest that EMMQ has a more powerful growth inhibitory effect than cisplatin by activating intrinsic pathway cell death in wild-type p53 NSCLC cells.. In this work using NSCLC cells, we showed that the interference of ΔΨm in EMMQ-treated cells appeared not only in A549 cells, but also in H1299 cells with stable expression of ectopic p53 (Fig.7A and 9B). However, ΔΨm was not affected in p53-null H1299 and H1299/p53R267P cells (Fig.7A and 9B). The results showed that EMMQ-induced cell apoptosis are depends on the status of p53 through permeabilization of the mitochondria and release of cytochrome c into the cytoplasm (Fig. 7B and 11B). However, release of cytochrome c was not affected in p53-null H1299 and H1299/p53R267P cells (Fig. 11B). The results suggest that p53 may trigger permeabilization of the outer mitochondrial membrane through direct activation of proapoptotic Bcl-2 proteins Bax or Bak or through binding and inactivating of antiapoptotic Bcl-2 proteins such as Bcl-2 or Bcl-XL [67-69]. The interference of ΔΨm and inactivating of antiapoptotic Bcl-2 proteins led to release of cytochrome c [70-73].. 32.

(36) Activation of p53 can occur in response to a number of cellular stresses, including DNA damage, hypoxia and nucleotide deprivation. Several forms of DNA damage have been shown to activate p53, including those generated by ionizing radiation (IR), radio-mimetic drugs, ultraviolet light (UV) and chemicals such as methyl methane sulfonate [29]. In the present study, we confirmed that EMMQ has cytotoxic effects on A549 and H460 cells, and we also demonstrated that EMMQ induced DNA damage (Fig. 3) and activation of p53 in vitro and vivo (Fig. 6, and 8) and all the effects were dose-dependent. Our data also showed that EMMQ induced cell cycle arrest in G2/M at 24 h and apoptosis at 48 h in A549 and H460 cells (Fig. 4 and 5). In the present study, we confirmed that the sensitivities of EMMQ can be offset by knocking-down p53 (Fig. 12) and the final apoptotic cell death reduced (Fig. 13). On the other hand, activate of p53 reduced expression levels of Bcl-2 and Akt [74]. Our study confirmed that EMMQ reduced levels of Akt that were reverted by knocking down p53 (Fig. 13). These results suggested EMMQ-mediated apoptosis in the presence of p53.. The PI3K/Akt pathway is a key signaling pathway involved in cell proliferation and differentiation, and is activated in many cancers [75]. More accumulating evidences suggested that signaling through PI3K/Akt is critically involved in trophoblast invasion and migration [76, 77]. Activation of PI3K/Akt pathways regulated of cell adhesion molecules E-cadherin and β-catenin, contributing to the attenuation of cell-cell adhesion and promoting the enhanced motility and migration [78]. The 33.

(37) induction of EMT by nuclear β-catenin has been explored during development in many cell lines and tumors [79]. Our data showed that the Akt and β-catenin level were decreased as the concentrations of EMMQ were increased in A549 cells (Fig. 14A). Fig. 14A demonstrated that EMMQ inhibited cell-cell adhesion and EMT by decreasing β-catenin expression.. MMP plays an important role in tumorigenesis, including early carcinogenesis events, tumor growth and tumor invasion and metastasis. Cell surface localization and activation of MMP is essential for cells to migrate, through rearrangement of ECM to suit cell migration. Certain MMP, such as gelatinases and membrane type 1 MMP, have special mechanisms to localize at leading edges in both types of cell migration [80, 81]. The growth inhibition in H460 and A549 cells was between 8 and 10µM. The data demonstrated that EMMQ inhibited metastasis with concentration of less than 5 µM in in A549 and H460 cells (Fig 14) by migration assay. The results suggested that EMMQ-inhibited metastasis may not be caused by apoptosis. More experiments are needed to confirm the drug effectiveness in metastasis. 34.

(38) VII. CONCLUSION This study supports the role of a small molecule indolylquinoline as a novel regulator against NSCLC. The sensitivity and specificity of EMMQ against NCSLC dells depend on the status of p53by activating intrinsic mitochondrial pathway (Fig. 15). This study extends the feasibility of the use of EMMQ as cancer therapeutics specific against malignant cells, and more extensive exploration on the underlying mechanisms that control the function of the targeted p53 pathway should provide broadened scope prior to clinical application of the drug. This therapeutic approach with EMMQ may have the potential implication and new alternative to overcome limits of current available therapies.. 35.

(39) VIII.. FIGURE AND LEGENDS. (A). (B). 36.

(40) (C). Figure 1 The structure of a new synthetic compound EMMQ and its inhibitory effect on cell growth of wild type p53 NSCLC cell lines. (A) The chemical structure of EMMQ. (B) Sensitivity of H460 cells to cisplatin. The cells were cultured in a 96-well plate for 48 h, and then were treated with different concentrations of cisplatin as indicated. After 48 h of treatment, MTT cell viability assay was performed and the blue formazan dye produced by the viable cells was quantified by measuring the absorbance of 540 nm after elution by DMSO. The DMSO treated cells were served as 100% control. Data are represented as the mean values ± standard deviation (SD). The asterisk ( ) p< 0.05 and (. )p<. 0.01 indicated a significant difference from the vehicle control. (C) Treatment with various concentrations of EMMQ in A549, H1299, H460 and MRC-5 cells. Symbol (D) indicates DMSO. 37.

(41) (A). (B). 38.

(42) Figure 2 EMMQ treatment inhibits growth of wild type p53 NSCLC cell lines. (A) Colony formation assay. The seeded cells in 6-well plates were treated with 5, 8 and 10 μM of EMMQ, respectively, for 48 h. The media were replaced with fresh media and incubated at 37 °C. After 24 days, the cells were fixed and stained with 10% methylene blue in 70% ethanol for counting colony numbers. (B) Statistical analysis of colony formation. The numbers of colonies of DMSO treated cells were served as 100% control. Data are presented as the mean values ± standard deviation (SD). The asterisk ( ) p< 0.05 indicates a significant difference from the vehicle control. Symbol (D) indicates DMSO.. 39.

(43) (A). (B). 40.

(44) Figure 3 EMMQ treatment induces DNA damage in A549 and H460 cells. (A) Representative profiles of comet assay. EMMQ- induced DNA damage in human NSCLC cells were examined by comet assay. The A549, H1299 and H460 cells were seeded in 12-well plates and incubated with various concentrations of EMMQ for 24 h. The cells were collected and DNA damage was determined by comet assay as described in the Materials and Methods. (B) Statistical analysis of comet assay. The asterisk (. ) p< 0.01 and (. ) p< 0.001 indicates a significant. difference from the vehicle control. Symbol (D) indicates DMSO.. 41.

(45) (A). 42.

(46) (B). 43.

(47) Figure 4 EMMQ treatment induces change in cell cycle distribution in A549 and H460 cells. (A) A549, H1299 and H460 cells were treated with various concentrations of EMMQ for 48 h. Cells were stained with PI and detected the cell cycle distribution by flow cytometry. The populations of cells in sub-G1 phase increased on EMMQ treatment and the populations were at the expenses of G1 cells. (B) A549 cells treated with EMMQ resulted in a significant increase in the proportion of cells at the G2 phase at 8 μM for 24 h. Symbol (D) indicates DMSO.. 44.

(48) (A). (B). 45.

(49) Figure 5 Apoptosis induced by EMMQ in NSCLC cell line. (A) Apoptosis assay: A549, H1299 and H460 cells were treated with EMMQ for 48 h and analyzed by flow cytometry after double staining with Annexin V-FITC/PI. The top right quadrant indicates late apoptosis and the bottom right early apoptosis. (B) The quantitative determination of apoptosis cell population. Early (dark) and late (light) apoptotic cell populations are shown for different cell lines. Symbol (D) indicates DMSO.. 46.

(50) (A). (B). Figure 6 EMMQ treatment induces apoptotic cell death in intrinsic pathway related proteins in A549 and H460 cells. (A) The protein lysates of the collected A549, H1299 and H460 cells treated with 5, 8 and 10 μM of EMMQ for 48 h were analyzed by Western blot as specified in Materials and Methods. (B) Cells treated with 5 μM EMMQ were collected at different time points (12, 24 and 48 h) and were analyzed by Western blot. Symbol (D) indicates DMSO.. 47.

(51) (A). (B). 48.

(52) Figure 7 EMMQ treatment causes apoptosis through reduced ΔΨm and enhanced cytochrome c release in A549 and H460 cells. (A) Evaluation of ΔΨm following EMMQ treatment. Cells treated with 5, 8 and 10 μM of EMMQ were evaluated for changes in ΔΨm in both A549 (8 and 12 h) and H1299 cells (12 h). (B) The induced release of cytochrome c from mitochondria to cytosol in A549 and H460 cells treated with 2 or 5 μM EMMQ or DMSO vehicle control for 24 h. The cells were stained with Mitotracker Green (mitochondrial staining), DAPI (blue, nuclear staining) and antibody conjugated with TRITC for cytochrome c. The pointed arrow signified the co-localization of red color cytochrome c and green color mitochondria. Symbol (D) indicates DMSO.. 49.

(53) (A). (B). 50.

(54) (C). (D). 51.

(55) (E). Figure 8 EMMQ inhibits growth of xenograft tumors by A549 cells in nude mice. (A) The mice were injected subcutaneous with EMMQ (at a dosage of 1 mg/kg/mouse) or vehicle control twice a week for four consecutive weeks. The pointed arrow signified the dosage of injection. The mean tumor volumes were measured at the time points of drug administration as specify. The statistics were the results of three independent experiments ( p < 0.01, compared with tumors that received vehicle control). (B) The average body weights of mice showed no significant difference as compared with those treated with PBS. (C) The picture of the dissected tumors treated with EMMQ in mice. The picture showed that EMMQ inhibited tumor growth as compared with PBS. (D) Statistical analysis of the dissected tumors treated with EMMQ in mice. The horizontal lines represent mean values of the tumor sizes at harvest ( p < 0.05). (E) Protein lysates from the tumors in mice treated with PBS and EMMQ were analyzed by Western blot.. 52.

(56) (A). (B). 53.

(57) Figure 9 EMMQ treatment causes apoptosis through reduced ΔΨm on H1299 cells with stable expression of ectopic p53. (A) The cell viability in H1299 cells with stable expression of ectopic WTp53 (H1299/p53) and p53R267P (H1299/p53R267P) treated with different concentrations of EMMQ for 48 h and the viability were determined by MTT assay. (B) The ΔΨm changes were determined in H1299/p53 and H1299/p53R267P cells treated with 5, 8 and 10 μM of EMMQ for 12 h. Symbol (D) indicates DMSO.. 54.

(58) 55.

(59) Figure 10 EMMQ treatment induces change in cell cycle distribution on H1299 cells with stable expression of ectopic p53. Both H1299/p53 and H1299/p53R267P cells were incubated with different concentrations (5, 8 and 10 μM) of EMMQ for 48 h followed by cell cycle determination after being stained with PI. Symbol (D) indicates DMSO.. 56.

(60) (A). (B). 57.

(61) Figure 11 EMMQ treatment causes apoptosis through enhanced cytochrome c release on H1299 cells with stable expression of ectopic p53. (A) Quantitative determination of cells of early (dark) and late (light) apoptosis in H1299/p53 and H1299/p53R267P cells after being incubated with different concentrations of EMMQ or DMSO for 48 h. Cells were doubly stained with Annexin V-FITC and PI and then analyzed by flow cytometry. (B) EMMQ treatment causes apoptosis by enhancing cytochrome c release in both H1299/p53 and H1299/p53R267P treated with 5 μM EMMQ or vehicle control DMSO for 24 h. The pointed arrow signified the co-localization of red color cytochrome c and green color mitochondria, while blue color stood for nucleus. Symbol (D) indicates DMSO.. 58.

(62) (A). (B). 59.

(63) Figure 12 EMMQ treatment on p53 silencing in A549 and H460 cells proliferation. (A) A549 cells were transfected with either p53 shRNA or NS, incubated for 18 h and then treated with 10 μM EMMQ for 48 h. The cell numbers were counted by trypan blue exclusion assay. ( ) p<0.05, compared with drug alone and NS RNA-transfected cells. Symbol (-) indicates no transfection. (B) Similar experiments performed in H460 cells. Symbol (D) indicates DMSO.. 60.

(64) (A). (B). Figure 13 EMMQ treatment reduces apoptotic cell death in intrinsic pathway related proteins on p53 silencing in A549 and H460 cells. (A) Expression of p53 and apoptosis markers in shRNA and NS transfected cells as monitored by western blot. (B) Similar experiments were performed in H460 cells. Symbol (-) indicates no transfection. 61.

(65) (A). (B). 62.

(66) (C). 63.

(67) (D). Figure 14 EMMQ inhibits cell migration of A549, H1299 and H460 cells. (A) Protein lysates of A549, H1299 and H460 cells treated with different concentrations (5, 8 and 10 μM) of EMMQ for 48 h. (B) The photographs of wound closures were captured in a dose-dependent manner showing inhibition of migration by treating with EMMQ for 48 h. (C) Statistical analysis of wound area. Determine the length of the scratched area wound at 0, 24 and 48 h respectively. Percent closure of difference concentration (%) = 100 × [width (0, 24 and 48 h) /width (0 h)]. The asterisk (. ) p< 0.01 and (. ) p< 0.001 indicated a significant. difference from the vehicle. (D) EMMQ reduced MMP-2 and MMP-9 activity of A549 cells in a dose-dependent manner. The MMP activities in cultured media were determined by gelatin zymography and identified by clear zones of digested gelatin. Symbol (D) indicates DMSO. 64.

(68) Figure 15 Proposed mechanism for EMMQ-induced apoptotic cell death in NSCLS cells. In this work, we demonstrated that EMMQ activates p53 and inhibits cell growth in human NSCLC cells. The compound causes DNA damage, decreases ΔΨm and accelerates apoptosis in cells harboring wild type p53. The effects were substantiated by the onset of apoptosis through p53 activation after treatment. Thus, p53 status may serve as a vital determinant in EMMQ-mediated apoptosis through mitochondria pathway by affecting Bcl-2 expression, cytochrome c release and cleavage of PARP.. 65.

(69) Part Ⅱ I. ABSTRACT Human liver cancer is the fifth most frequently diagnosed cancer worldwide. The important limitation in the clinical battle against this tumor is its marked intrinsic and acquired refractivity to the available chemotherapies. Anticancer agents effective against chemo-resistant cells are greatly needed for liver cancer treatment. Previously, our study have identified EMMQ as an effective drug in the treatment of lung cancer cells in vitro and in animal models. In this work, results show EMMQ treatment may inhibit cell growth and induce apoptosis in HCC. EMMQ induced apoptosis in HCC cells with wild-typed p53, and is less potent in cells with mutant p53 and in p53-null. The study also demonstrated that EMMQ induces apoptosis through two major pathways. First, the compound induced cell death through the intrinsic pathway by first damaging DNA increasing expression of p53 and γ-H2AX and decreasing cyclin D1 and CDK 2, finally leading to G1 arrest in HepG2 cells as studied cell cycle. Second, the tumor suppressor gene p53 was activated following a reduction of ΔΨm, ROS generation and down-regulating Akt, Bcl-2, Bax, cytochrome c, caspase-3 and cleavage of PARP, the critical events leading to cell death in HepG2 cells treated with EMMQ. The in vitro findings indicate that EMMQ is a promising candidate for the treatment of liver cancer.. Keywords: Liver cancer, p53, apoptosis. 66.

(70) II. 中文摘要肝癌是世界排名第五大常見的癌症。臨床顯示治療這些肝癌腫瘤中的重要的限制是治療過程中化療藥物長久投藥後的失效且肝癌對這些藥物產生抗藥性的問題。因此，開發肝癌治療中所產生化療失效與抗藥性的抗癌藥物是迫切需要的。之前我們已經確認 EMMQ 於肺癌細胞與活體的模式中是有效治療的效果。在此研究中證明 EMMQ 可抑制肝癌細胞的細胞生長進而誘導細胞凋亡。 EMMQ 誘導的細胞凋亡為 wild type p53 的肝細胞癌（HCC）細胞，但是對 mutant p53 和 p53-null 細胞不敏感。數據顯示此化合物以內源性的途徑方式誘導細胞死亡。研究證明了 EMMQ 通過兩個主要途徑誘導細胞凋亡。此化合物使 HepG2 細胞的 DNA 損傷進而活化 p53 和 γ-H2AX，降低 cyclin D1 和 CDK 2 的表現，導致細胞週期於 G1 期停滯。其次，此化合物使腫瘤抑制基因 p53 活化，干擾粒線體膜電位，使得 ROS 產生， Akt 與 Bcl-2 表現降低，Bax 和細胞色素 c 的釋放，讓 caspase-3 和 PARP 裂解。細胞實驗的結果證明，在肝癌的治療上，EMMQ 是一個有潛力的抗癌藥物。關鍵字: 肝癌, p53, EMMQ, 細胞凋亡. 67.

(71) III. INTRODUCTION 1. Liver cancer Liver cancer is the fifth most frequently diagnosed cancer in men and the ninth most common cancer in women, and it is the second most frequent cause of cancer related death worldwide [1, 82]. HCC, a primary malignancy of the hepatocyte, accounts for 85% to 90% of all primary liver cancer [83]. Platinum- analogue agents have been widely-used chemotherapeutic drugs for HCC treatment over the past 30 years [84]. Unfortunately, the intrinsic or acquired drug resistance mainly accounts for the partial responses in HCC patients to platinum analogue regimen [85, 86]. Platinum-based analogs have been developed, including cisplatin and carboplatin, which also act by forming DNA crosslinks and significantly attenuate the growth of tumors, but their use is followed by the development of chemo-resistance as well as cytotoxicity [87]. Many lines of clinical investigation indicate that none of the adjuvant therapies is particularly effective in treating HCC after surgery and systemic traditional chemotherapy has a very low response rate for HCC [88]. Therefore, new effective and well-tolerated therapy strategies are urgently needed.. 2. Aims of study Herein, this study is to determine whether EMMQ induces apoptosis through DNA damage pathway of intrinsic apoptosis in HCC cell lines. The study used the HCC cell lines Huh7, HepG2 and Hep3B, which have mutant p53, wild type p53 and null p53, respectively, to determine 68.

(72) EMMQ reduced cell viabilities began with DNA damage followed by arresting of cell population at G1 phase in HepG2 cells. The study further evaluated mitochondrial membrane potential suppression followed by ROS and cytochrome c expression in HepG2 cells. The development of apoptosis depended on the DNA damage whose activation leads to the status of tumor suppressor p53, cleavage of PARP and caspase-3 in HepG2 cells. Furthermore, we reported that transfection of shRNA of p53 suppressed DNA damage and apoptosis effect of drug sensitivity. Thus, in view of the constant need to seek new active drugs against liver cancer with the least adversity, the EMMQ provides a potential alternative to treat human liver cancer cells by repressing growth of cells and p53 modulates the effects.. 69.

(73) IV. MATERIALS AND METHODS 1. Cell culture and reagent HCC cell lines, HepG2 (HB-8065, wild-type p53) and Hep3B (HB-8064, p53-null) were acquired from American Type Culture Collection (Manassas, VA). Huh7 (mutant p53) cells were from Japanese Collection of Research Bioresources. Normal human liver cell line LO2 was purchased from the Chinese Academy of Science Committee Type Culture Collection Cell Bank. All cells were cultured in DMEM supplemented with L-glutamine, sodium pyruvate and 10% FBS at 37 °C in a humidified atmosphere with 5% CO2. The HCC cell lines and human hepatic cells were treated with EMMQ at 1, 5 and 10 μM. All measurements were conducted in duplicate in three independent experiments.. 2. Cell growth assay Cell growth inhibition or cell number was determined by measuring MTT dye absorbance or by trypan blue cell counting. A total of 3 × 103cells per well were seeded in 96-well microtiter plates for an MTT assay. Cells were allowed to attach overnight, and then treated with different concentrations of EMMQ for 48 h at 37 °C. After removing the supernatant, formazan crystals were dissolved in 100 μL DMSO and the absorbance were measured at 570 nm. The inhibitory concentration at 50% growth (IC50) was determined.. 3. Colony forming assay 70.

(74) Cells were seeded at 500 cells per well in a 12-well plates for 16 h to allow attachment. The cells were treated with EMMQ at different concentrations or vehicle control for 48 h at 37°C and 5% CO2 in a humid environment. After 10 days, the plates were washed twice in PBS, fixed with paraformaldehyde, stained with 0.05% crystal violet, washed with PBS and air dried. The size and number of stained colonies with more than 50 cells were counted under inverted phase contrast microscope. Colony formation was calculated as a percentage to untreated control cultures.. 4. Comet assay The HCC cells were cultured in 12-well plates at a density of 1 × 105 cells/well and incubated with various concentrations of EMMQ for 3, 6, 12 and 24 h. After incubation, cells were harvested and mixed with low melting point agarose at 37 °C. This mixture were placed on the top of 0.5% agarose (normal melting point) on the slide, and then covered with a covership until solid. Subsequently, the covership was removed gently and some agarose were added onto this slide, and then covered with the covership again. The slide was placed until the mixture was solid, and put in chilled alkaline lysis buffer for electrophoresis. Afterwards, the slide was gently washed with neutralized buffer. The DNA was stained with PI. The tails were observed under a fluorescence microscope and quantified by using CometScore™ software.. 5. Determination of apoptosis 71.

(75) Flow cytometry analysis of cell cycle Cells were treated with EMMQ for 48 h and collected by trypsin and 3,000 rpm centrifugation for 5 min. The cell pellet was suspended with 70% ethanol at -20 °C overnight, washed with PBS, then incubated with 10 µg/mL RNase A and 10 µg/mL of PI for 20 min staining in the dark at 37 °C. Flow cytometry was used for detection. Data were plotted and analyzed by using software FlowJo.. Double staining with Annexin V-FITC and PI Cells were seeded at 1 ×105 cells per well in 12-well plates and treated by various concentrations of EMMQ incubated at 37 °C for 48 h. The cells were trypsinized and stained with1 μL annexin-V/FITC (20 µg/mL, BD Bioscience) and 1 μL of PI (50 µg/mL) at 37 °C for 30 min in the dark. The early and late phase of apoptosis was detected by annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience). The flow cytometer FACS CaliburTM (BD Bioscience) was used for analysis. Data were analyzed using the FlowJo software.. 6. Measurement of intracellular ROS The intracellular ROS was detected by staining the cells with DCF-DA. The HCC cells were cultured in 12-well plates at a density of 1 × 105 cells/well and incubated with various concentrations of EMMQ for 6 and 24 h. Cells were incubated with 10 μM DCF-DA for 30 min at 37 °C. Cells were washed twice with PBS (pH 7.4), and the fluorescence intensity was recorded by flow cytometer FACS CaliburTM (BD 72.