In this dissertation novel non-leaving groups were employed to synthesize platinum complexes which can contribute to the understanding or improvement of anticancer action. These complexes basically consist of (NS)-chelate and amineplatinum complexes. Bidentate (NS)-donor ligands were used as non-leaving ligands in the syntheses of platinum(II) complexes with iodide, chloride, bromide and oxalate anions as leaving groups. These complexes were synthesized and studied since many questions regarding the interaction of sulfur donors and platinum still exists. These relate to thermodynamic and kinetic factors and their influence on anticancer action. In this dissertation the properties of novel platinum(II) complexes of a bidentate ligand having an aromatic nitrogen-donor atom in combination with a thioethereal sulfur atom capable of forming a five membered ring with platinum(II) were studied. The general structure of the (NS) -ligands used were N-alkyl-2-methylthioalkyl imidazole. Alkyl groups used were methyl, ethyl and propyl. Although amine complexes of platinum have been extensively studied there are some new aspects of these that are worthwhile investigating. In this dissertation amines having planar attachments which will be at an angle with the coordination plane viz. benzylamine and amines having cyclic aliphatic groups namely cyclopropyl and cyclohexyl were investigated. Some of the (NS) - and amineplatinum(II) complexes were oxidised to their mononitroplatinum(IV) analogues . The motivation for the synthesis of these complexes was the greater kinetic stability of platinum(IV) and recent research has shown that a specific type of platinum(IV) compound shows suitable properties as an anticancer agent. These complexes were characterised by a variety of spectral means (IR, NMR, mass spectroscopy) as well as elemental analysis, solubility determinations, thermal analysis (TGA), ionization studies and finally their anticancer behaviour towards three different cell lines(Hela, MCF 7, Ht29) and in this process they were compared to the behaviour of cisplatin as a reference. A few have shown promising anticancer behaviour.

The aim in this thesis, was to synthesize novel platinum coordination compounds, in order to develop compounds with improved anticancer action which could lead to an improved understanding of the mechanism by which they operate and at the same time, improve synthetic methods for their products. The initial work included the development of a novel synthetic method for 1R,2R-diaminocyclohexaneoxalato-platinum(II) (oxaliplatin), by using an essentially non-aqueous solvent medium and direct ligand exchange at elevated temperatures. This was done by a study of the kinetics of the reaction in a variety of conditions; such as relative reagent concentrations and ratios as well as solvent mixtures. An effective method was developed which could be applied industrially. An international patent was taken out on this method. Various amine complexes of platinum(II) were synthesized using chloro, bromo and oxalato groups as leaving groups. The non-leaving groups were selected having certain specific characteristics in mind. Novel mononitroplatinum(IV) complexes were synthesized, mostly with oxalato leaving groups. One of these in particular, had excellent anticancer behaviour. Another trichloromononitro complex was also synthesized with very good anticancer properties. Two international patents were filed for the latter two compounds. As far as possible, all compounds were studied by spectrometric, chromatographic and thermal methods. They were also tested against 3 cancer cell lines namely cervical (Hela), Colon (HT29) and Breast (MCF7) cancer cells.

<p>Since the discovery that cis-diamminedichloroplatinum (II) possessed great antitumor properties, researchers have been synthesizing and screening metal coordination complexes for their potential anti-tumor activity. Hundreds of compounds have been tested, and few compounds possessed anti-tumor activity. This paper examines how the physical and chemical properties of complexes of the platinum group metals affect their toxicity and anti-tumor activity.</p> / Master of Science

Over the past 200 years, much attention has been paid to natural products for their great contribution in the industry of drug development as many of them have been shown effective against various diseased conditions in humans by virtue of their structural diversity and biological potency. Therefore, they are undeniably a rich resource for the discovery of novel bioactive compounds. To date, many of the mainstay anticancer agents often lead to undesirable side effects and/or develop rapid emergence of drug resistance. Therefore, new therapeutic remedies are desperately needed. In fact, many active compounds are derived from macrocyclic natural products. The identification of their molecular targets may assist researchers to synthesize biological agents for combating particular diseased conditions. Cycloheptapeptides that modulate specific molecular pathways in suppressing the proliferation of cancer cells are potential candidates for anticancer therapeutics and/or chemopreventive agents. In the current research project, we have demonstrated that MV-A, a novel cycloheptapeptide with the unique amino acid DMCPA isolated from Maytenus variabilis (Loes.) C. Y. Cheng (Celastraceae), showed potent cytotoxic activities against a panel of human cancer cell lines, and is worthy for further investigation. Objectives--The objectives of this study were to i) evaluate the anticancer effect, ii) elucidate the mechanism of action, and iii) identify the binding target(s) of the natural cycloheptapeptide MV-A. Methods--We first carried out various kinds of cellular and animal studies for validating the in vitro and in vivo anticancer efficacy of MV-A. Next, we performed a number of bioassays to ascertain the inhibitory effect of MV-A on several major cancer-associated pathways, including apoptosis, cell cycle arrest, senescence and metastasis. The biochemical assays included sulforhodamine B colorimetric assay, flow cytometric analyses of apoptosis and cell cycle arrest, Western blotting, real-time polymerase chain reactions (qPCR) arrays, senescence-associated β-galactosidase staining, phospho-specific protein arrays, as well as migration and invasion staining experiments. Lastly, we also identified the potential protein targets of MV-A by biochemical means, particularly the drug affinity responsive target stability (DARTS) approach. Results--MV-A is a potent anti-proliferative agent against a variety of cancer cells. It inhibited the proliferation of the human colorectal carcinoma (CRC) HCT116 cells with an IC50 value of 2.28 nM. However, the application of MV-A at 2.68 nM did not induce significant apoptosis; rather it caused a notable cell-cycle arrest at the G1 phase. Moreover, the treatment with this compound (0.68 to 2.68 nM) led to a remarkable senescence in cancer cells as well as a mitigated cellular migration. Meanwhile, the expression levels of some components of the p16 cascade and PI3K-AKT pathway, so as several epithelial-to-mesenchymal transition (EMT) molecules were suppressed by MV-A. Furthermore, HSP90, calnexin, EF2, 14-3-3 and annexin A1 were identified as the direct binding targets of MV-A in our DARTS analysis.Conclusions--In the present study, our results indicated that the novel cycloheptapeptide MV-A inhibited proliferation and migration of CRC HCT116 cells via the induction of cellular senescence and modulation of multiple pathways, including the p16/Rb, PI3K-AKT and EMT signaling pathways. These results revealed a potential role of MV-A in cancer therapy. The direct binding targets of MV-A further uncovered its molecular actions against different diseased conditions. Our findings strongly support the development of MV-A as a therapeutic agent for combating cancerous pathologies, explicitly CRC.

Over the past 200 years, much attention has been paid to natural products for their great contribution in the industry of drug development as many of them have been shown effective against various diseased conditions in humans by virtue of their structural diversity and biological potency. Therefore, they are undeniably a rich resource for the discovery of novel bioactive compounds. To date, many of the mainstay anticancer agents often lead to undesirable side effects and/or develop rapid emergence of drug resistance. Therefore, new therapeutic remedies are desperately needed. In fact, many active compounds are derived from macrocyclic natural products. The identification of their molecular targets may assist researchers to synthesize biological agents for combating particular diseased conditions. Cycloheptapeptides that modulate specific molecular pathways in suppressing the proliferation of cancer cells are potential candidates for anticancer therapeutics and/or chemopreventive agents. In the current research project, we have demonstrated that MV-A, a novel cycloheptapeptide with the unique amino acid DMCPA isolated from Maytenus variabilis (Loes.) C. Y. Cheng (Celastraceae), showed potent cytotoxic activities against a panel of human cancer cell lines, and is worthy for further investigation. Objectives--The objectives of this study were to i) evaluate the anticancer effect, ii) elucidate the mechanism of action, and iii) identify the binding target(s) of the natural cycloheptapeptide MV-A. Methods--We first carried out various kinds of cellular and animal studies for validating the in vitro and in vivo anticancer efficacy of MV-A. Next, we performed a number of bioassays to ascertain the inhibitory effect of MV-A on several major cancer-associated pathways, including apoptosis, cell cycle arrest, senescence and metastasis. The biochemical assays included sulforhodamine B colorimetric assay, flow cytometric analyses of apoptosis and cell cycle arrest, Western blotting, real-time polymerase chain reactions (qPCR) arrays, senescence-associated β-galactosidase staining, phospho-specific protein arrays, as well as migration and invasion staining experiments. Lastly, we also identified the potential protein targets of MV-A by biochemical means, particularly the drug affinity responsive target stability (DARTS) approach. Results--MV-A is a potent anti-proliferative agent against a variety of cancer cells. It inhibited the proliferation of the human colorectal carcinoma (CRC) HCT116 cells with an IC50 value of 2.28 nM. However, the application of MV-A at 2.68 nM did not induce significant apoptosis; rather it caused a notable cell-cycle arrest at the G1 phase. Moreover, the treatment with this compound (0.68 to 2.68 nM) led to a remarkable senescence in cancer cells as well as a mitigated cellular migration. Meanwhile, the expression levels of some components of the p16 cascade and PI3K-AKT pathway, so as several epithelial-to-mesenchymal transition (EMT) molecules were suppressed by MV-A. Furthermore, HSP90, calnexin, EF2, 14-3-3 and annexin A1 were identified as the direct binding targets of MV-A in our DARTS analysis.Conclusions--In the present study, our results indicated that the novel cycloheptapeptide MV-A inhibited proliferation and migration of CRC HCT116 cells via the induction of cellular senescence and modulation of multiple pathways, including the p16/Rb, PI3K-AKT and EMT signaling pathways. These results revealed a potential role of MV-A in cancer therapy. The direct binding targets of MV-A further uncovered its molecular actions against different diseased conditions. Our findings strongly support the development of MV-A as a therapeutic agent for combating cancerous pathologies, explicitly CRC.

No description available.

The novel diterpenoid taxol isolated from the western yew Taxus brevifolia is one of the most important lead compounds to emerge from the search for anticancer agents from plants. It shows consistent clinical activity against ovarian cancer and may also be active against other cancers. In this study, the preparation of various taxol derivatives was investigated, with the objective of better understanding the structural requirements for activity in the taxol series. The 7-hydroxyl group of taxol was derivatized with a photoaffinity label and other reagents as a beginning of the project to understand the interaction of taxol and tubulin, and the activity of all the derivatives in a tubulin assay was determined. A study of the deacylation and reacylation reactions of baccatin 111 was carried out in order to find conditions suitable for the preparation of 2-debenzoylbaccatin Ill , and thus 2-debenzoyltaxol. Finally, the reactions of taxol with various electrophilic reagents were investigated, and the structures of products with an opened oxetane ring and/or contracted ring A were determined. Biological assay results are reported on many of the compounds in this investigation. / Ph. D.

by Joyce Chui Kwan Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 61-75). / Abstracts in English and Chinese. / Acknowledgments --- p.i / List of Abbreviations --- p.iii / Abstract / English --- p.1 / Chinese --- p.2 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Tumor Formation --- p.3 / Chapter 1.2 --- Anti-tumor Pathways --- p.4 / Chapter 1.3 --- Aim of Project --- p.13 / Chapter Chapter 2 --- The In Vivo effect of Polysaccharopeptide / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and Methods --- p.17 / Chapter 2.3 --- Results --- p.18 / Chapter 2.4 --- Discussion --- p.19 / Chapter Chapter 3 --- Cytotoxicity / Chapter 3.1 --- Introduction --- p.23 / Chapter 3.2 --- Materials and Methods --- p.26 / Chapter 3.3 --- Results --- p.28 / Chapter 3.4 --- Discussion --- p.28 / Chapter Chapter 4 --- Anti-angiogenic Effect / Chapter 4.1 --- Introduction --- p.30 / Chapter 4.2 --- Materials and Methods --- p.35 / Chapter 4.3 --- Results --- p.39 / Chapter 4.4 --- Discussion --- p.42 / Chapter Chapter 5 --- Immunomodulation / Chapter 5.1 --- Introduction --- p.45 / Chapter 5.2 --- Materials and Methods --- p.47 / Chapter 5.3 --- Results --- p.50 / Chapter 5.4 --- Discussion --- p.52 / Chapter Chapter 6 --- General Discussion --- p.57 / References --- p.61

Kong Ka-lai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 126-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.ix / List of Figures/Table --- p.xiii / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- An Overview on Cancer --- p.1 / Chapter 1.2 --- Biological Effects of Chlorophyllin --- p.7 / Chapter 1.2.1 --- CHL as Photosensitizer --- p.7 / Chapter 1.2.2 --- CHL as Antioxidant --- p.8 / Chapter 1.2.3 --- CHL as Anticarcinogenic Agent --- p.9 / Chapter 1.3 --- Regulation of Cell Cycle --- p.13 / Chapter 1.3.1 --- Cell-Cycle Checkpoints --- p.13 / Chapter 1.3.2 --- Cell-Cycle Regulatory Proteins --- p.15 / Chapter 1.4 --- Regulation of Mitogen-Activated Protein Kinase (MAPK) Signaling Cascade --- p.21 / Chapter 1.5 --- Programmed Cell Death (or Apoptosis) --- p.27 / Chapter 1.5.1 --- Regulation of Caspase-Dependent Apoptosis --- p.28 / Chapter 1.5.2 --- Regulation of Caspase-Independent Cell Death --- p.32 / Chapter 1.5.3 --- Bcl-2 Family Proteins in Modulation of Cell Death --- p.32 / Chapter 1.6 --- In Vivo Antitumor Screening System --- p.37 / Chapter 1.7 --- Aims of the Present Study --- p.38 / Chapter Chapter 2 --- In Vitro Studies of the Anticancer Effect of Chlorophyllin / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.1.1 --- DNA-Flow Cytometric Analysis --- p.51 / Chapter 2.1.2 --- Western Blot Analysis --- p.54 / Chapter 2.2 --- Materials and Methods --- p.56 / Chapter 2.2.1 --- Maintenance of Cell Lines --- p.56 / Chapter 2.2.2 --- Cytotoxic and Cytostatic Effects on the Cancer Cells --- p.56 / Chapter 2.2.3 --- DNA-Flow Cytometric Analysis --- p.60 / Chapter 2.2.4 --- Western Blot Analysis --- p.61 / Chapter 2.2.5 --- JC-1 Mitochondrial Potential Sensor --- p.64 / Chapter 2.2.6 --- Caspase Inhibitors --- p.65 / Chapter 2.2.7 --- Statistical Analysis --- p.66 / Chapter 2.2.8 --- Densitometric Analysis --- p.66 / Chapter 2.3 --- Results --- p.67 / Chapter 2.3.1 --- Effects of CHL on the Growth of Human Cancer Cells by MTT Assay --- p.67 / Chapter 2.3.2 --- Effect of CHL on the Proliferation of MCF-7 Cells by Chemi-BrdU Incorporation --- p.69 / Chapter 2.3.3 --- Effect of CHL on Cell Cycle of MCF-7 Cells --- p.71 / Chapter 2.3.4 --- Effect of CHL on the Cyclin D1 Expression in MCF-7 Cells --- p.74 / Chapter 2.3.5 --- Effects of CHL on JNK and c-Jun Expressions and Their Phosphorylations in MCF-7 Cells --- p.76 / Chapter 2.3.6 --- Effect of CHL on DNA fragmentation in MCF-7 Cells --- p.78 / Chapter 2.3.7 --- Effect of CHL on Mitochondrial Membrane Potential of MCF-7 Cells --- p.80 / Chapter 2.3.8 --- Effects of CHL on the PARP Expression and Cleavage in MCF-7 Cells --- p.83 / Chapter 2.3.9 --- "Effects of CHL on Bcl-2, Bcl-xL and Bad Expressions in MCF-7 Cells" --- p.85 / Chapter 2.3.10 --- Effects of CHL on Caspase Activations in MCF-7 Cells --- p.88 / Chapter 2.3.11 --- Effects of Caspase Inhibitors on the CHL-Induced Apoptosis in MCF-7 Cells --- p.90 / Chapter 2.4 --- Discussion --- p.93 / Chapter Chapter 3 --- In Vivo Studies of the Anticancer Effect of Chlorophyllin / Chapter 3.1 --- Introduction --- p.104 / Chapter 3.2 --- Materials and Methods --- p.106 / Chapter 3.2.1 --- Transplantation of MCF-7 Cells into the Nude Mice and Treatment --- p.106 / Chapter 3.2.2 --- Western Blot Analysis --- p.107 / Chapter 3.2.3 --- Statistical Analysis --- p.107 / Chapter 3.3 --- Results --- p.108 / Chapter 3.3.1 --- In Vivo Antitumor Activity of CHL --- p.108 / Chapter 3.3.2 --- In Vivo Effects of CHL on Cyclin D1 and Bcl-2 Expressions in MCF-7 Solid Tumor --- p.111 / Chapter 3.4 --- Discussion --- p.113 / Chapter Chapter 4 --- General Discussion --- p.115 / References --- p.126

神經母細胞瘤是一種交感神經系統的腫瘤。它是最常見的兒童顱外實體瘤。神經母細胞瘤約佔兒童腫瘤的8-10%，佔15%的兒童腫瘤死亡率。目前神經母細胞瘤的治療方法包括外科手術， 化學藥物治療， 放射治療， 幹細胞移植， 誘導分化治療和免疫治療。 然而，這些治療方法通常會導致許多無法避免的嚴重的副作用。因此，開發能高效抑制神經母細胞瘤但對正常細胞無明顯副作用的新型藥物顯得至關重要。最近，用來源於天然產物或中藥的化合物治療癌症引起了科學家的廣泛關注。靛玉紅-3’-肟（Indirubin-3’-oxime, I3M）和色胺酮（tryptanthrin）分別是從板藍根中分離得到的靛藍生物鹼和吲哚喹唑啉類生物鹼。據研究報導，這兩種生物鹼具有多種生物學和藥理學作用，包括抗菌，抗炎症和抗腫瘤作用。它們對體外的多種人腫瘤細胞具有抗腫瘤作用。然而，它們對人神經母細胞瘤的調節作用和作用機理仍不太清楚。在我的博士研究課題中，我們對板藍根生物鹼包括靛玉紅-3’-肟和色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了研究和探討。 / 首先，我們研究了靛玉紅-3’-肟對人神經母細胞瘤的抗腫瘤活性和作用機制。實驗結果表明，靛玉紅-3’-肟能夠抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，並且其抑制效果呈時間和濃度依賴性。然而，靛玉紅-3’-肟對正常細胞無顯著的細胞毒性作用。對其生長抑制作用機制的研究結果表明靛玉紅-3’-肟能夠特異性地減少LA-N-1細胞系中線粒體的調節子ERR和 PGC-1的表達，從而導致線粒體生成減少，線粒體膜電位降低以及線粒體活性氧（ROS）增加。靛玉紅-3’-肟還增加週期蛋白依賴性蛋白激酶（CDK）抑制蛋白p27{U+1D37}{U+2071}{U+1D56}¹的蛋白水平並降低週期蛋白依賴性蛋白激酶2（CDK2）和細胞週期蛋白E（Cyclin E）的表達，從而導致細胞週期阻滯在G0/G1期。 另外，我們發現靛玉紅-3’-肟能減少SH-SY5Y細胞系的線粒體生成，增加細胞內活性氧的水準從而導致細胞週期停滯在G0/G1期和細胞凋亡。以上結果表明靛玉紅-3’-肟可能通過破壞線粒體的功能從而導致LA-N-1和SH-SY5Y細胞的細胞週期阻滯和誘導SH-SY5Y細胞的細胞凋亡來發揮其抗腫瘤的作用。 / 接著，我們對色胺酮對人神經母細胞瘤的抗腫瘤活性和作用機制進行了探討。我們研究的結果表明，色胺酮可以時間和濃度依賴性地抑制人神經母細胞瘤LA-N-1, SH-SY5Y 和 SK-N-DZ細胞系的生長，而對正常的細胞無顯著的細胞毒性。對色胺酮抑制人神經母細胞瘤生長的機制研究表明，色胺酮能顯著地降低細胞週期蛋白（Cyclin D1和 Cyclin D3）和週期蛋白依賴性蛋白激酶（CDK4和CDK6）的蛋白水平從而導致細胞週期停滯在G0/G1期。色胺酮可以激活半胱氨酸天冬氨酸蛋白酶8,9和3/7（caspase 8, caspase 9 和 caspase 3/7）從而誘導LA-N-1細胞凋亡。色胺酮還可以誘導LA-N-1細胞分化，表現為神經細胞分化的細胞形態，乙醯膽鹼酯酶活性的增加和多種細胞分化的分子標記的表達上調。另外，色胺酮還能降低LA-N-1細胞中N-myc的表達。有趣的是，通過RNA干擾技術降低N-myc的表達能誘導LA-N-1細胞的分化。總的來說，以上結果顯示色胺酮通過誘導細胞週期阻滯，細胞凋亡和細胞分化從而發揮其抗腫瘤的作用。它可能被開發為治療有N-myc基因擴增的高危的人神經母細胞瘤的潛在藥物。 / 此外，我們還研究了靛玉紅-3’-肟和色胺酮是否具有抗血管生成的作用。體外實驗的研究結果表明，靛玉紅-3’-肟和色胺酮能夠濃度依賴性地抑制人微血管上皮細胞 （HMEC-1細胞）的增殖，遷徙和血管生成，但對HMEC-1細胞卻沒有顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮能顯著地抑制小鼠體內的基質膠栓（Matrigel plug）的血管生成。對它們抑制血管生成的機制的研究表明，靛玉紅-3’-肟能下調血管生成素1（Ang-1）和基質金屬蛋白酶2（MMP2）的表達，上調血管生成素2（Ang-2）的表達。靛玉紅-3’-肟能結合到血管內皮生長因數受體2（VEGFR2） 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和下游的MEK/ERK和PI3K/AKT/GSK信號轉導通路。色胺酮同樣可以抑制多種血管生成因子（Ang-1,PDGFB 和MMP2）的表達。此外，它可以結合到血管內皮生長因數受體2 的ATP結合位點上從而抑制血管內皮生長因數受體2的磷酸化和血管內皮生長因數受體2介導的ERK1/2信號通路。以上的體外和體內實驗研究結果表明靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。它們可能被開發為治療血管生成相關疾病的潛在藥物。 / 總而言之，我們的研究結果表明靛玉紅-3’-肟和色胺酮通過誘導人神經母細胞瘤細胞的細胞週期阻滯，細胞凋亡或誘導神經細胞分化從而抑制人神經母細胞瘤細胞的生長。然而，它們對正常細胞無顯著的細胞毒性作用。此外，靛玉紅-3’-肟和色胺酮通過靶向血管內皮生長因數受體2介導的信號通路來發揮其抗血管生成的作用。未來的研究將進一步探討靛玉紅-3’-肟和色胺酮對人神經母細胞瘤細胞的分子作用機理。另外，通過人神經母細胞瘤細胞的裸鼠移植瘤動物模型可進一步去了解這些板藍根生物鹼在體內的抗腫瘤效果。 / Neuroblastoma, a tumor of the sympathetic nervous system, is the most common extracranial solid cancer in childhood. It accounts for 8% to 10% of all childhood cancers and for approximately 15% of cancer deaths in children. Current treatment modalities consist of surgery, chemotherapy, radiation therapy, stem cell transplantation, differentiation therapy and immunotherapy. However, these treatments often cause severe and inevitable side effects. It is important to develop novel drugs with higher efficacy on neuroblastoma cells and minimal side effects on normal cells. The use of new promising therapeutic compounds derived from natural products or Chinese herbs have attracted much attention of scientist as an alternative strategy in cancer treatment. Indirubin-3’-oxime (I3M) is an indigo alkaloid and tryptanthrin is an indoloquinazoline alkaloid which can be isolated from the dried roots of medicinal indigo plants known as Banlangen. These two alkaloids have been reported to possess various biological and pharmacological activities, such as anti-microbial, anti-inflammatory, and anti-tumor effects. They were found to exhibit potent anti-tumor activities on various types of human cancer cells in vitro. However, their modulatory effects on human neuroblastoma and the underlying mechanisms remain poorly understood. In my PhD project, the possible anti-tumor activities and action mechanisms of Banlangen alkaloids, including I3M and tryptanthrin, on human neuroblastoma cells were investigated. / Firstly, the anti-cancer effects of I3M on human neuroblastoma cells and the underlying mechanisms were investigated. I3M was found to inhibit the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies showed that I3M specifically decreased the expression of mitochondrial regulators ERRγ and PGC-1βand resulted in decreased mitochondrial mass and altered mitochondrial function characterized by reduction in mitochondrial membrane potential and elevation of reactive oxygen species (ROS) level in LA-N-1 cells. I3M also increased the level of cyclin-dependent kinase (CDK) inhibitor p27{U+1D37}{U+2071}{U+1D56}¹ and reduced the levels of CDK2 and cyclin E in LA-N-1 cells, leading to cell cycle arrest at the G0/G1 phase. In addition, I3M was also found to reduce the mitochondrial mass and increase the ROS level leading to cell cycle arrest at G0/G1 phase and apoptosis in SH-SY5Y cells. These results, when taken together, suggest that I3M might exert its anti-tumor activity by causing mitochondrial dysfunction which led to cell cycle arrest in LA-N-1 cells and resulted in cycle arrest and apoptosis in SH-SY5Y cells. / The anti-tumor effects and action mechanisms of tryptanthrin on the human neuroblastoma cells were also examined. Our results showed that tryptanthrin inhibited the growth of the human neuroblastoma LA-N-1, SH-SY5Y and SK-N-DZ cells in a concentration- and time-dependent manner, but exhibited little, if any, direct cytotoxicity on normal cells. Mechanistic studies indicated that tryptanthrin significantly reduced the protein levels of cyclin D1, cyclin D3, CDK4 and CDK6 leading to cell cycle arrest at G0/G1 phase. In addition, tryptanthrin activated caspase 8, caspase 9 and caspase 3/7 resulting in apoptosis of the human neuroblastoma LA-N-1 cells. Moreover, tryptanthrin induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Tryptanthrin treatment also led to the significant reduction of N-myc expression in LA-N-1 cells. Interestingly, down-regulating N-myc expression using siRNA induced neuronal differentiation of LA-N-1 cells. Collectively, these results indicate that tryptanthrin might exert its anti-tumor activity on the human neuroblastoma LA-N-1 cells by inducing cell cycle arrest, apoptosis and neuronal differentiation. It might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification. / Moreover, the anti-angiogenic activities of I3M and tryptanthrin were studied. Our results showed that I3M and tryptanthrin inhibited the proliferation, migration, and tube formation of the human microvascular endothelial HMEC-1 cells in vitro in a concentration-dependent manner but exhibited no significant cytotoxicity on these cells. Moreover, I3M and tryptanthrin markedly suppressed the in vivo angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that I3M down-regulated the expression of Ang-1 and MMP2 and up-regulated the expression of Ang-2. It also bound to the ATP-binding site of VEGFR2 and inhibited the phosphorylation of VEGFR2 leading to suppression of the down-stream MEK/ERK and PI3K/AKT/GSK signaling pathways in HMEC-1 cells. Similarly, tryptanthrin also reduced the expression of several angiogenic factors (Ang-1, PDGFB and MMP2) in HMEC-1 cells. In addition, tryptanthrin also bound to the ATP-binding site of VEGFR2 and suppressed the phosphorylation of VEGFR2 and VEGFR2-mediated ERK1/2 signaling pathway in HMEC-1 cells. Collectively, our results demonstrated that I3M and tryptanthrin exhibited anti-angiogenic activity both in vitro and in vivo by specifically targeting the VEGFR2-mediated signaling pathways and might be exploited as potential therapeutic candidates for the treatment of angiogenesis-related diseases. / In conclusion, our findings indicate that I3M and tryptanthrin might exert their growth-inhibitory effect on the human neuroblastoma cells by causing cell cycle arrest, inducing apoptosis or inducing neuronal differentiation. However, they exhibited minimal cytotoxicity towards the normal cells. Moreover, I3M and tryptanthrin were found to possess anti-angiogenic activities by targeting the VEGFR2-mediated signaling pathways. In the future, investigations should be focused on further elucidation of the molecular action mechanisms of I3M and tryptanthrin on human neuroblastoma cells and to test the anti-tumor efficacy of I3M and tryptanthrin in animal models, using human neuroblastoma xenografts in nude mice. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liao, Xuemei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 206-229). / Abstract also in Chinese. / Acknowledgments --- p.i / Abbreviations --- p.ii / Publications --- p.vi / Abstract --- p.vii / 摘要 --- p.xii / Table of Contents --- p.xvi / Chapter One / General Introduction --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- Epidemiology of neuroblastoma --- p.2 / Chapter 1.1.2 --- Classification of neuroblastoma --- p.6 / Chapter 1.1.3 --- Clinical symptoms and diagnosis of neuroblastoma --- p.10 / Chapter 1.1.4 --- Molecular pathogenesis of neuroblastoma --- p.13 / Chapter 1.1.4.1 --- Genetic alterations in neuroblastoma --- p.13 / Chapter 1.1.4.2 --- Disruption of cell division cycle, apoptotic and signaling pathways --- p.16 / Chapter 1.1.5 --- Treatment strategies --- p.19 / Chapter 1.1.5.1 --- Low-risk neuroblastoma treatment strategy --- p.19 / Chapter 1.1.5.2 --- Intermediate-risk neuroblastoma treatment strategy --- p.20 / Chapter 1.1.5.3 --- High-risk neuroblastoma treatment strategy --- p.21 / Chapter 1.1.5.4 --- Side effects of treatment --- p.23 / Chapter 1.2 --- Banlangen alkaloids --- p.23 / Chapter 1.2.1 --- Overview of Banlangen alkaloids --- p.23 / Chapter 1.2.2 --- Biological and pharmacological effects of Banlangen alkaloids --- p.28 / Chapter 1.2.2.1 --- Anti-inflammatory activity --- p.28 / Chapter 1.2.2.2 --- Anti-microbial activity --- p.29 / Chapter 1.2.2.3 --- Anti-tumor activity --- p.30 / Chapter 1.2.2.4 --- Other biological activities --- p.32 / Chapter 1.2.3 --- Bioavailability of Banlangen alkaloids --- p.33 / Chapter 1.2.4 --- Toxicity of Banlangen alkaloids --- p.34 / Chapter 1.3 --- Aims and scope of this project --- p.36 / Chapter Two / Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Animals --- p.39 / Chapter 2.1.2 --- Cell lines --- p.39 / Chapter 2.1.3 --- Cell culture media --- p.41 / Chapter 2.1.4 --- Drugs and chemicals --- p.42 / Chapter 2.1.5 --- Reagents and buffers for cell culture --- p.44 / Chapter 2.1.6 --- General staining solutions --- p.47 / Chapter 2.1.7 --- Reagents and buffers for cell growth assays --- p.48 / Chapter 2.1.8 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.9 --- Reagents and buffers for acetylcholine esterase activity assay --- p.50 / Chapter 2.1.10 --- Reagents and buffers for immunocytochemistry --- p.51 / Chapter 2.1.11 --- Reagents and buffers for total RNA extraction --- p.53 / Chapter 2.1.12 --- Reagents and buffers for reverse transcription --- p.54 / Chapter 2.1.13 --- Reagents for quantitative real-time polymerase chain reaction (qRT-PCR) --- p.56 / Chapter 2.1.14 --- Reagents and buffers for Western blotting --- p.59 / Chapter 2.1.15 --- Assay kits --- p.65 / Chapter 2.2 --- Methods --- p.68 / Chapter 2.2.1 --- Culture of cells --- p.68 / Chapter 2.2.2 --- MTT assay --- p.69 / Chapter 2.2.3 --- Cell proliferation assay --- p.70 / Chapter 2.2.4 --- Trypan blue exclusion test --- p.70 / Chapter 2.2.5 --- Cytotoxicity assay --- p.71 / Chapter 2.2.6 --- Colony-forming assay --- p.72 / Chapter 2.2.7 --- Cell cycle analysis --- p.72 / Chapter 2.2.8 --- Assessment of apoptosis --- p.73 / Chapter 2.2.9 --- Caspase activity determination --- p.74 / Chapter 2.2.10 --- Mitochondrial mass assay --- p.75 / Chapter 2.2.11 --- Reactive oxygen species (ROS) assay --- p.75 / Chapter 2.2.12 --- Mitochondrial membrane potential determination --- p.76 / Chapter 2.2.13 --- Morphological detection of cell differentiation --- p.76 / Chapter 2.2.14 --- Acetylcholine esterase activity determination --- p.77 / Chapter 2.2.15 --- Immunocytochemistry --- p.77 / Chapter 2.2.16 --- RNA interference --- p.78 / Chapter 2.2.17 --- Wound healing assay --- p.79 / Chapter 2.2.18 --- Tube formation assay --- p.79 / Chapter 2.2.19 --- In vivo Matrigel plug assay --- p.80 / Chapter 2.2.20 --- Phospho-VEGFR2 Sandwich ELISA assay --- p.80 / Chapter 2.2.21 --- Isolation of total cellular RNA --- p.81 / Chapter 2.2.22 --- Reverse transcription (RT) --- p.82 / Chapter 2.2.23 --- Quantitative real-time PCR --- p.83 / Chapter 2.2.24 --- Total protein extraction --- p.84 / Chapter 2.2.25 --- Protein concentration determination --- p.84 / Chapter 2.2.26 --- Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) --- p.85 / Chapter 2.2.27 --- Semi-dry Western blotting --- p.85 / Chapter 2.2.28 --- Enhanced chemiluminescence (ECL) assay --- p.87 / Chapter 2.2.29 --- Molecular docking --- p.87 / Chapter 2.2.30 --- Statistical analysis --- p.88 / Chapter Three / Modulatory effects and action mechanisms of indirubin-3'-oxime on human neuroblastoma cells --- p.89 / Chapter 3.1 --- Introduction --- p.90 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Indirubin-3’-oxime inhibited the growth and colony formation of human neuroblastoma cells in vitro --- p.94 / Chapter 3.2.2 --- Indirubin-3’-oxime exhibited no significant cytotoxicity on normal cells --- p.101 / Chapter 3.2.3 --- Indirubin-3’-oxime induced G0/G1 cell cycle arrest in LA-N-1 cells --- p.103 / Chapter 3.2.4 --- Indirubin-3’-oxime caused mitochondrial dysfunction in LA-N-1 cells --- p.106 / Chapter 3.2.5 --- Indirubin-3’-oxime selectively reduced ERR γ and PGC-1β protein and mRNA levels in LA-N-1 cells --- p.111 / Chapter 3.2.6 --- Indirubin-3’-oxime induced cell cycle arrest at G0/G1 phase and apoptosis of SH-SY5Y cells --- p.113 / Chapter 3.2.7 --- Indirubin-3’-oxime reduced mitochondrial mass and elevated mitochondrial ROS level in SH-SY5Y cells --- p.115 / Chapter 3.2.8 --- Indirubin-3’-oxime increased the caspase 8, caspase 9 and caspase 3/7 activities in SH-SY5Y cells --- p.117 / Chapter 3.3 --- Discussion --- p.119 / Chapter Four / Modulatory effects and action mechanisms of tryptanthrin on human neuroblastoma cells --- p.125 / Chapter 4.1 --- Introduction --- p.126 / Chapter 4.2 --- Results --- p.129 / Chapter 4.2.1 --- Tryptanthrin inhibited the cell growth and colony formation of human neuroblastoma cells --- p.129 / Chapter 4.2.2 --- Tryptanthrin exhibited no significant cytotoxicity on normal cells --- p.136 / Chapter 4.2.3 --- Tryptanthrin induced cell cycle arrest at G0/G1 phase --- p.138 / Chapter 4.2.4 --- Tryptanthrin induced apoptosis of LA-N-1 cells --- p.140 / Chapter 4.2.5 --- Tryptanthrin induced morphological neuronal differentiation in LA-N-1 cells --- p.143 / Chapter 4.2.6 --- Tryptanthrin induced the expression of neuronal differentiation markers --- p.146 / Chapter 4.2.7 --- Tryptanthrin down-regulated the expression of N-myc in LA-N-1 cells --- p.149 / Chapter 4.3 --- Discussion --- p.152 / Chapter Five / Anti-angiogenesis effects and action mechanisms of indirubin-3'-oxime and tryptanthrin --- p.158 / Chapter 5.1 --- Introduction --- p.159 / Chapter 5.2 --- Results --- p.163 / Chapter 5.2.1 --- Indirubin-3’-oxime and tryptanthrin inhibited the proliferation of endothelial cells --- p.163 / Chapter 5.2.3 --- Indirubin-3’-oxime and tryptanthrin reduced the tube formation of endothelial cells --- p.168 / Chapter 5.2.4 --- Indirubin-3’-oxime and tryptanthrin blocked angiogenesis in the in vivo Matrigel plug model --- p.171 / Chapter 5.2.5 --- Indirubin-3’-oxime and tryptanthrin reduced the angiogenic gene expression in endothelial cells --- p.174 / Chapter 5.2.6 --- Indirubin-3’-oxime and tryptanthrin attenuated VEGFR2-mediated signaling pathways in endothelial cells --- p.176 / Chapter 5.2.7 --- Indirubin-3’-oxime bound to the ATP-binding site of VEGFR2 kinase domain --- p.181 / Chapter 5.2.8 --- Tryptanthrin bound to the ATP-binding site of VEGFR2 kinase domain --- p.182 / Chapter 5.3 --- Discussion --- p.184 / Chapter Six / Conclusions and future perspectives --- p.191 / References --- p.206