Report (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 53-61). Also available via the Internet.

Triggering direct cytotoxicity has been the most common strategy for developing cancer treatments. The cytotoxic regimens currently used in the clinic mainly include radiation therapy, classic chemotherapeutic drugs (e.g. DNA damaging drugs and anti-mitotic drugs) and selected new targeted drugs. Although these therapies are the standard of care for most cancer patients, they suffer significant limitations: responses to these therapies vary significantly between cancer types and patients; sensitive cancers tend to acquire resistance; and they cause serious toxicity, particularly to dividing cells in the bone marrow and gut, and to neurons. It is not clear whether major improvements in cytotoxic anticancer therapies are possible; if they are, progress is likely to come from either new methods for identifying sub-populations of patients that respond well to current drugs, or developing new therapies with novel cytotoxic mechanisms. To pursue the above two avenues towards potential improvement of cytotoxic therapies, this thesis investigates: biomarkers that determine the sensitivity of distinct cancer cell types to common anti-mitotic chemotherapeutics; and the mechanistic basis to employ alternating electric field and Natural Killer cells as alternative methods to trigger cancer cell death. The study uses time-lapse microscopy as the major technique to characterize and quantify response dynamics to the different cytotoxic treatments, and the results provide important new insight not only for understanding existing cytotoxic anticancer drugs but also for developing novel cytotoxic regimens.

In this study, the synthesis of TTX by three species of TTX-producing bacteria (Vibrio alginolyticus, Microbacterium arabinogalactanolyticum and Serratia marcescens) was conducted in a 10-L fermentor under the same controlled fermentation conditions for each of a period of 60 hours. The bacterial growth curves were monitored and the TTX synthesized in the culture medium was determined by HPLC. The TTX biosynthesis was found limited at the microgram level per L of culture medium with toxicities 14.7 MU (mouse unit) and 13.0 MU per mL in the partially purified culture medium of V. alginolyticus and M. arabinogalactanolyticum respectively by mouse bioassay. In the studies on SW480 and SW620 colorectal carcinoma cell lines, the expression, distribution, invasion and proliferation of voltage-gated sodium channels (VGSCs) were investigated by MTT assay (24-48 hours) and wound healing assay (0-120 hours). The different subtypes of VGSCs were expressed by semiquantitative RT-PCR and the locations of Nav1.5 and Nav 1.7 were detected by immunofluorescence microscopy. In the MTT assay, 40μmol/L of TTX showed significant inhibitory effect on both cell lines, with maximum inhibition rate, 33% and 40%, in SW480 and SW620 respectively. In the wound-healing assay, the inhibitory rate of 80μmol/L of TTX on SW480 reached 22% after 120 hours, compared with 30% in the control group. Moreover, VGSCs were highly expressed in both SW480 and SW620, with the main subtypes of Nav1.5 and Nav1.7 located on the cell surface, which might increase the metastatic rate of the cell lines. Keywords: Tetrodotoxin (TTX), Bacterial synthesis, Anticancer, VGSCs

The aim of this thesis was to investigate the properties of novel platinum compounds with possible potential as anticancer agents, and to determine how their behaviour could lead to a better understanding of the chemistry involved. The final criteria were improvement of their anticancer behaviour. Since many questions are still unanswered as to the role of sulfur in anticancer action, studies were undertaken to synthesize novel platinum(II) complexes having non-leaving groups consisting of a combination of an aromatic nitrogen and thioetherial sulfur capable of forming a five membered ring upon coordination. The structural unit was 1-methyl-2-methylthioalkyl/aryl. Numerous complexes formed by these ligands each having chloro, bromo, iodo and oxalato leaving groups were then fully characterized. The results obtained by the various synthetic methods were compared and explained in terms of the chemistry involved. The role of the sulfur donor was indicated in both the halo- and oxalato-complexes and proved to be strongly influenced by the nature of the leaving groups. Their differences are reflected in their anticancer behaviour. The study was extended to mononitroplatinum(IV) complexes, in view of the kinetically stable platinum(IV) compounds and advantages related to this. A specific mononitroplatinum(IV) complex which proved to have good anticancer and STAT 3 properties could according to the literature not be synthesized successfully in a good yield and a high degree of purity. The results of extensive studies showed that the main problem centred around the simultaneous reactions in equilibrium during the synthesis. A number of these species formed as a result of side reactions could be identified and their close separation factors indicated chromatographically. The mechanism of these reactions and the unstable intermediate species involved could be rationalized and compared to analogues in the literature. All the complexes studied were characterized by spectral and thermal methods both in solution as well as the solid state. Their anticancer behaviour towards three anticancer cell lines (Hela, MCF 7, Ht 29) were determined and acted as a guide towards possible structural modifications for their improved capability. Three crystal structures of platinum(II) complexes were determined. The extent of the ionization of the platinum(II) complexes as well the redox potentials (Pt(II) / Pt(IV)) of the platinum(IV) complexes were particularly important factors pertaining to their anticancer action.

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The synthesis of 7-N-acetyldemethyllavendamycin butyl amide (47), 7-Nacetyldemethyllavendamycin isopropyl amide (48), 7-N-acetyldemethyllavendamycin amide of piperidine (49), 7-N-acetyldemethyllavendamycin amide of pyrrolidine (50), 7N-acetyldemethyllavendamycin amide of morpholine (51), demethyllavendamycin butyl amide (52), demethyllavendamycin amide of pyrrolidine (53), and demethyllavendamycin amide of morpholine (54) are described. Pictet Spengler condesation of 7-acetamido-2formylquinoline-5,8-dione (28) with tryptophan butyl amide (66), tryptophan isopropyl amide (67), tryptophan amide of piperidine (68), tryptophan amide of pyrrolidine (69), and tryptophan amide of morpholine (70) in an anisole - pyridine solution directly afforded the five lavendamycin amides 47-51. Compounds 52, 53, and 54 were obtained by hydrolysis of 47, 50, and 51 with 70% H2SO4-H20 solution.Aldehyde 28 was prepared according to the following general procedure.Nitration of 8-hydroxy-2-methylquinoline (30) yielded 8-hydroxy-2-methyl-5,7 dinitroquinoline (31). Compound 31 was then hydrogenated and acylated with acetic anhydride to yield 5,7-diacetamido-2-methyl-8-acetoxyquinoline (33). Compound 33 was oxidized by potassium dichromate to give 7-acetamido-2-methylquinoline-5,8-dione (27). Treatment of 27 with selenium dioxide in refluxing 1,4-dioxane afforded compound 28.Compounds 66, 67, 68, 69, and 70 were synthesized from compounds 61,62, 63, 64, and 65. These compounds were deprotected with ammonium formate in the presence of 10% Palladium on charcoal in methanol under an argon balloon at atmospheric pressure.Compounds 61, 62, 63, 64, and 65 were obtained from 58 with butylamine, isopropylamine, piperidine, pyrrolidine, and morpholine respectively in the presence of triethylamine under an argon balloon at atmospheric pressure.Compound 58 was synthesized by the reaction of N-carbobenzyloxytryptophan, with N-hydroxy succinimide, in the presence of N-dicyclohexylcarbodimide in dried and distilled dioxane under an argon balloon at atmospheric pressure.The structures of the novel compounds 58, 47, 48, 49, 50, 51, 52, 53, and 54 were confirmed by 1H NMR, IR, EIMS, and HRMS.The structures of protected and deprotected amides 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 were also confirmed by 1 H NMR and IR spectroscopy. / Department of Chemistry

by Lee Kin Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 214-225). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vii / ABSTRACT IN CHINESE --- p.ix / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xv / CONTENTS --- p.xvi / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Cordyceps --- p.2 / Chapter 1.1.1 --- Pharmacological Functions of Cordyceps --- p.5 / Chapter 1.1.1.1 --- Anti-tumor Activities --- p.5 / Chapter 1.1.1.2 --- Immunomodulatory Activities --- p.7 / Chapter 1.1.1.3 --- Hepatic Functions --- p.9 / Chapter 1.1.1.4 --- Cardiovascular Functions --- p.10 / Chapter 1.1.1.5 --- Renal Functions --- p.10 / Chapter 1.2 --- Biological Activities of Cordycepin --- p.12 / Chapter 1.2.1 --- Inhibition of RNA Synthesis --- p.12 / Chapter 1.2.2 --- Disruption of Microtubule Network --- p.12 / Chapter 1.2.3 --- Inhibition of Nucleic Acid Methylation --- p.13 / Chapter 1.2.4 --- Enhancement of Cell Differentiation --- p.13 / Chapter 1.2.5 --- Anti-tumor Activity --- p.13 / Chapter 1.2.6 --- Anti-fungal Activity --- p.14 / Chapter 1.3 --- Hepatocellular Carcinoma --- p.16 / Chapter 1.3.1 --- Incidence and Risk Factor of Hepatocellular Carcinoma --- p.16 / Chapter 1.3.2 --- Treatment of Hepatocellular Carcinoma --- p.16 / Chapter 1.3.2.1 --- Hepatic Resection --- p.16 / Chapter 1.3.2.2 --- Liver Transplantation --- p.17 / Chapter 1.3.2.3 --- Non-surgical Therapeutic Modalities for Hepatocellular Carcinoma --- p.17 / Chapter 1.3.3 --- Human Hepatocellular Carcinoma Cell Lines --- p.20 / Chapter 1.3.3.1 --- Human Hepatocellular Carcinoma Cell Line HepG2 --- p.20 / Chapter 1.3.3.2 --- Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.20 / Chapter 1.4 --- Multidrug Resistance of Tumor Cells --- p.22 / Chapter 1.4.1 --- Multidrug Resistance Mediated by P-Glycoprotein --- p.22 / Chapter 1.4.1.1 --- Location and Structure of P-Glycoprotein --- p.22 / Chapter 1.4.1.2 --- Substrates of P-Glycoprotein --- p.23 / Chapter 1.4.1.3 --- Mechanism of Action of P-Glycoprotein --- p.23 / Chapter 1.4.2 --- Reversal of Multidrug Resistance by Chemosensitizers --- p.24 / Chapter 1.5 --- Leukemia / Chapter 1.5.1 --- Acute Myeloid Leukemia --- p.28 / Chapter 1.5.2 --- Acute Promyelocytic Leukemia and Treatment --- p.28 / Chapter 1.5.3 --- Human Promyelocytic Leukemia Cell Lines --- p.30 / Chapter 1.5.3.1 --- HL-60 --- p.30 / Chapter 1.5.3.2 --- NB-4 --- p.30 / Chapter 1.6 --- Objectives of Study --- p.33 / Chapter 1.6.1 --- Study of Biological Activities of Cordyceps militaris --- p.33 / Chapter 1.6.2 --- Study of Anti-tumor Activity of Cordycepin --- p.33 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.34 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Animal --- p.35 / Chapter 2.1.2 --- Cell Culture --- p.35 / Chapter 2.1.2.1 --- Cell Lines --- p.35 / Chapter 2.1.2.2 --- Cell Culture Media --- p.37 / Chapter 2.1.2.3 --- Buffers and other Reagents --- p.38 / Chapter 2.1.3 --- Reagents and Buffers for Different Assays --- p.40 / Chapter 2.1.3.1 --- Reagents and Buffers for Flow Cytometry --- p.40 / Chapter 2.1.3.2 --- Reagents and Buffers for DNA Fragmentation Assay --- p.40 / Chapter 2.1.3.3 --- Reagents and Buffers for Western Blot Analysis --- p.42 / Chapter 2.1.3.4 --- Reagents and Buffers for Caspases Activities --- p.46 / Chapter 2.1.3.5 --- Reagents and Buffers for Cell Surface Marker (CD3，CD4 and CD8) Staining --- p.48 / Chapter 2.1.3.6 --- Reagents and Buffers for Cytokine Determination --- p.49 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Preparation of Water Extract of Cordyceps militaris --- p.50 / Chapter 2.2.2 --- MTT Assay --- p.50 / Chapter 2.2.3 --- In Vivo Anti-tumor Study --- p.51 / Chapter 2.2.4 --- Preparation of Splenic Lymphocytes --- p.51 / Chapter 2.2.5 --- Lymphoproliferation Test --- p.51 / Chapter 2.2.6 --- "Cell Surface Marker (CD3, CD4 and CD8) Staining" --- p.52 / Chapter 2.2.7 --- Measurement of Cytokine Production by ELISA --- p.53 / Chapter 2.2.8 --- In Vivo Study of the Toxicity of WECM --- p.54 / Chapter 2.2.9 --- Cell Cycle Analysis --- p.55 / Chapter 2.2.10 --- DNA Fragmentation Assay --- p.56 / Chapter 2.2.11 --- Cell Morphology Study --- p.57 / Chapter 2.2.12 --- Detection of Apoptotic Cells with Annexin V-FITC/PI --- p.57 / Chapter 2.2.13 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent Dye --- p.58 / Chapter 2.2.14 --- Simultaneous Detection of Mitochondrial Membrane Potential and Intracellular Hydrogen Peroxide --- p.58 / Chapter 2.2.15 --- Western Blot Analysis --- p.59 / Chapter 2.2.15.1 --- Total Protein Extraction --- p.59 / Chapter 2.2.15.2 --- Determination of Protein Amount --- p.59 / Chapter 2.2.15.3 --- SDS Polyacrylamide Gel Electrophoresis --- p.60 / Chapter 2.2.15.4 --- Electroblotting of Protein --- p.61 / Chapter 2.2.15.5 --- Probing of Proteins with Antibodies --- p.61 / Chapter 2.2.15.6 --- Enhanced Chemiluminescence (ECL) Assay --- p.64 / Chapter 2.2.15.7 --- Extraction of Cytosolic Protein --- p.64 / Chapter 2.2.16 --- Determination of Caspases Enzymatic Activity --- p.65 / Chapter 2.2.16.1 --- Extraction of Proteins --- p.65 / Chapter 2.2.16.2 --- Determination of Caspase-3 Activity --- p.65 / Chapter 2.2.16.3 --- Determination of Caspase-8 Activity --- p.66 / Chapter 2.2.16.4 --- Determination of Caspase-9 Activity --- p.67 / Chapter 2.2.17 --- Hemolysis Assay --- p.69 / Chapter 2.2.18 --- Measurement of Intracellular Doxorubicin Accumulation --- p.69 / Chapter CHAPTER 3: --- ANTI-TUMOR AND IMMUNO- MODULATORY EFFECTS OF cordyceps militaris --- p.71 / Chapter 3.1 --- In Vitro Anti-tumor Study of Water Extract of Cordyceps militaris (WECM) --- p.72 / Chapter 3.2 --- In Vitro Study of Immunomodulatory Effect of WECM --- p.78 / Chapter 3.3 --- In Vivo Anti-tumor Study of WECM --- p.80 / Chapter 3.4 --- Anti-tumor Effect of WECM Mediated by Stimulating T-cell Proliferation --- p.83 / Chapter 3.5 --- Toxicity Studies of WECM --- p.92 / Chapter CHAPTER 4: --- ANTI-PROLIFERATIVE EFFECT OF THE ACTIVE COMPONENTS OF cordyceps militaris --- p.97 / Chapter 4.1 --- "Anti-proliferative Study of D-mannitol, Adenosine and Cordycepin (3'deoxyadenosine)" --- p.98 / Chapter 4.2 --- Anti-proliferative Study of Doxorubicin --- p.105 / Chapter 4.3 --- Accumulation of Doxorubicin in HepG2 and R-HepG2 Cells --- p.109 / Chapter 4.4 --- Cytotoxicity Study of Cordycepin and Doxorubicin on Normal Liver Cells --- p.114 / Chapter 4.5 --- Hemolytic Study of Cordycepin --- p.116 / Chapter CHAPTER 5: --- MECHANISTIC STUDY OF CORDYCEPIN IN THE INDUCTION OF APOPTOSIS IN LEUKEMIA CELLS --- p.118 / Chapter 5.1 --- Cell Cycle Analysis of Leukemia Cells --- p.119 / Chapter 5.2 --- Hallmarks of Apoptosis --- p.123 / Chapter 5.2.1 --- Induction of Phosphatidylserine Externalization in Leukemia Cells by Cordycepin --- p.123 / Chapter 5.2.2 --- Induction of DNA Fragmentation in Leukemia Cells by Cordycepin --- p.127 / Chapter 5.2.3 --- Morphological Changes in Leukemia Cells Induced by Cordycepin --- p.130 / Chapter 5.2.4 --- Caspase-3 Activation in Leukemia Cells Induced by Cordycepin --- p.133 / Chapter 5.3 --- Study of the Underlying Mechanisms of Cordycepin-induced Apoptosis in Leukemia Cells --- p.140 / Chapter 5.3.1 --- Induction of Mitochondrial Membrane Depolarization in Leukemia Cells --- p.140 / Chapter 5.3.2 --- Elevation of Intracellular Hydrogen Peroxide Level in Cordycepin-treated Leukemia Cells --- p.144 / Chapter 5.3.3 --- Induction of Cytochrome c Release from Mitochondria of Leukemia Cells --- p.148 / Chapter 5.3.4 --- Caspase-9 Activation in Leukemia Cells Induced by Cordycepin --- p.150 / Chapter 5.3.5 --- Involvement of Bcl-2 Family Members in Cordycepin-induced Apoptosis --- p.153 / Chapter 5.3.6 --- Involvement of Death Receptor Pathway in Cordycepin-induced Apoptosis in Leukemia Cells --- p.159 / Chapter CHAPTER 6: --- MECHANISTIC STUDY OF CORDYCEPIN IN THE INDUCTION OF CELL CYCLE ARREST IN HEPATOCELLULAR CARCINOMA CELLS --- p.164 / Chapter 6.1 --- Cell Cycle Analysis of Hepatocellular Carcinoma Cells --- p.165 / Chapter 6.2 --- Expression of Cell Cycle Regulatory Proteins in Cordycepin-treated Hepatocellular Carcinoma Cells --- p.170 / Chapter 6.3 --- Increased Expression of p21 in Cordycepin-treated Hepatocellular Carcinoma Cells --- p.176 / Chapter 6.4 --- Involvement of p53 in G2/M Phase Arrest of the Cell Cycle in Hepatocellular Carcinoma Cells --- p.178 / Chapter 6.5 --- Induction of Apoptosis in Cordycepin-treated R-HepG2 cells --- p.180 / Chapter CHAPTER 7: --- DISCUSSION --- p.185 / Chapter 7.1 --- In Vitro and In Vivo Studies in the Biological Activities of WECM --- p.186 / Chapter 7.2 --- Induction of Apoptosis in Leukemia Cells by Cordycepin --- p.192 / Chapter 7.3 --- Induction of Cell Cycle Arrest in Hepatocellular Carcinoma Cells by Cordycepin --- p.202 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.210 / REFERENCES --- p.214