A 24-hour fermentation of the optimized synbiotic composed of B. longum and EPR was performed to give a cell-free fermentation broth (S24). S24 was co-cultured with two colon cancer cell lines (Caco-2 and SW620) and normal colon cells (FHC). S24 significantly inhibited cell proliferation for both colon cancer cells but promoted FHC cell growth by 10-25% as shown by MTT and BrdU arrays. Primary DNA damage analysis by alkaline comet assay showed S24 caused DNA damage to a comparable extent as the positive control of 10 mM H2O2 (treated for 1 hour) for both cancer cells. Dynamic analysis on DNA damage-associated DNA repair showed the two colon cancer cells had different response pattern to S24. Flow cytometric analysis showed that both Caco-2 and SW620 when treated with S24 (IC 50=3.66 mM of acetate) were arrested initially at G2/M and subsequently at S phase accompanied with large sub-G1 peaks. Dual staining with PI/AnnexinV further proved the appearance of apoptosis. Live cell imaging analysis on Caco-2 cells treated with S24 showed the following events: mitochondria were rapidly destroyed within the first two-hour treatment, the cells bubbled and the nucleus condensed after the mitochondrial had shrunken, followed by apoptosis. / Despite active research on synbiotic on anti-carcinogenesis of colon cancer by synbiotics, the underlying mechanism still remains unclear. This study investigated a novel synbiotic composed of non-digestible carbohydrates (NDCs) extracted from mushroom sclerotia as prebiotics and Bifidobacteria as probiotics. Preliminary results on incubation of two probiotics ( Bifidobacterium longum and Lactobacillus brevis) and one pathogenic bacterium (Clostridium celatum) separately with 3 NDCs extracted from mushroom sclerotia [Poria cocos (PC), Polyporus rhinocerus (PR) and Pleurotus tuber-regium (PT)] indicated that the growth of B. longum and L. brevis was stimulated more preferentially than C. celatum after 72-hour fermentation. The short-chain fatty acid (SCFA) profile was dominated by acetate (> 98% of total SCFAs) with very little butyrate (< 2.0% of total SCFAs) and the organic matter disappearance (OMD) during fermentation was consistent with the bacterial growth. Among the synbiotic combinations, NDC from PR and B. longum gave the largest amount of acetate (2.47+/-0.232 mmol/g of organic matter disappearance). / Results obtained from human pathway finder RT2 Profiler(TM) PCR Array indicated that S24 could modulate the proliferation of colon cancer cells mainly by various pathways such as cell cycle and DNA damage repair, apoptosis and cell senescence, etc. In SW620 cells, PCR Array of Human Cell Cycle further revealed that the modulated genes mainly belonged to the gene cluster of S phase and DNA replication as well as G2 and G2/M transition. While for Caco-2 cells, the cell-cycle modulated genes mainly belonged to the cluster of G2 and G2/M transition. Immuno-blotting on the pivotal upstream regulators showed that phosphorylation of ATM at Serine 1981 was significantly increased in both cancer cells. Site-specific phosphorylation of pRB was decreased and phosphorylation of Chk1 was increased in both cancer cells while Chk2 were increased in SW620 cells. Cdc25A was phosphorylated at serine17 in both cancer cells. It can be proposed that the blockage of DNA synthesis or DNA damage was due to the down-regulation of some pivotal DNA replication related proteins such as RPA3, PCNA and MCMs, detected by ATM-Chk1/Chk2-Cdc25A pathway. This would cause the prolonged staying of cells at the G1/S checkpoint which further moved on to S phase arrest for SW620 cells. Moreover the sharply up-regulated p21, an important inhibitor of Cdk2 would further hinder the cells passing the G1/S checkpoint in SW620 cells. / The tumor suppressor p53 was detected phosphorylated at various sites in SW620 but not in Caco-2 cells. In SW620 cells, G2/M arrest was caused by the inhibition of CDK1/CDC2 due to increased expression of GADD45A and p21 and phosphorylation of Cdc25A, while for Caco-2, the G2/M arrest was caused by degradation of Cdc25A due to the absence of p53-activated GADD45A and p21 expression as shown in the pathway finder results. Some apoptosis-related proteins of Bax, Apaf-1 and PARP were modulated as shown by immuno-blotting in both colon cancer cells. (Abstract shortened by UMI.) / Gao, Shane. / Adviser: Peter Chi-Keung Cheung. / Source: Dissertation Abstracts International, Volume: 72-11, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 55-94). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Leung, Winnie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 155-164). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / Abbreviations --- p.xii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Clinical management of Cancer --- p.2 / Chapter 1.2 --- Multidrug resistance --- p.8 / Chapter 1.3 --- Aim of study --- p.14 / Chapter Chapter 2 --- Identification of gene contributing to multidrug resistance in human uterine sarcoma cells --- p.16 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Material and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Cell lines --- p.20 / Chapter 2.2.1.2 --- "Cell culture medium, supplements and buffers" --- p.20 / Chapter 2.2.1.3 --- Gene expression assay reagents --- p.22 / Chapter 2.2.1.4 --- Western blotting reagents --- p.24 / Chapter 2.2.1.5 --- MTT assay reagents --- p.29 / Chapter 2.2.1.6 --- Apoptosis analysis by flow cytometry reagents --- p.29 / Chapter 2.2.2 --- Metho --- p.ds / Chapter 2.2.2.1 --- Cell Culture --- p.31 / Chapter 2.2.2.2 --- MTT assay --- p.32 / Chapter 2.2.2.3 --- Gene expression essay (RT-PCR) --- p.33 / Chapter 2.2.2.4 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.37 / Chapter 2.2.2.5 --- Quantification of doxorubicin uptake by flow cytometry --- p.40 / Chapter 2.2.2.6 --- Apoptosis analysis by flow cytometry --- p.41 / Chapter 2.3 --- Results --- p.4 / Chapter 2.3.1 --- Cytotoxicity of doxorubicin on SA and DX5 cells --- p.43 / Chapter 2.3.2 --- mRNA expression of multidrug resistance related genes in SA and DX5 cells --- p.46 / Chapter 2.3.3 --- P-glycoprotein expression in SA and DX5 cells --- p.49 / Chapter 2.3.4 --- Doxorubicin (Dox) uptake by SA and DX5 cells --- p.51 / Chapter 2.3.5 --- Doxorubicin induced Apoptosis in SA and DX5 cells --- p.54 / Chapter 2.4 --- Discussion --- p.61 / Chapter 2.5 --- Conclusion --- p.65 / Chapter Chapter 3 --- Alternation in P-glycoprotein expression in DX5_Ninjl cells --- p.66 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials / Chapter 3.2.1.1 --- Cell lines --- p.70 / Chapter 3.2.1.2 --- "Cell culture medium, supplements and buffers" --- p.70 / Chapter 3.2.1.3 --- Gene expression assay reagents --- p.70 / Chapter 3.2.1.4 --- Western blotting reagents --- p.72 / Chapter 3.2.1.5 --- Plasmid DNA extraction --- p.75 / Chapter 3.2.1.6 --- Transient transfection --- p.76 / Chapter 3.2.1.7 --- MTT reagents --- p.76 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell culture --- p.78 / Chapter 3.2.2.2 --- Gene expression essay (RT-PCR) --- p.79 / Chapter 3.2.2.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.81 / Chapter 3.2.2.4 --- DNA plasmid extraction --- p.83 / Chapter 3.2.2.5 --- Transient transfection --- p.84 / Chapter 3.2.2.6 --- MTT assay --- p.85 / Chapter 3.2.2.7 --- Quantification of doxorubicin (Dox) uptake by flow cytometry --- p.86 / Chapter 3.3 --- Results / Chapter 3.3.1 --- mRNA expression of Ninjurinl (Ninj1) in SA and DX5 cells --- p.87 / Chapter 3.3.2 --- The protein expression of Ninjurinl (Ninj1) in SA and DX5 cells --- p.89 / Chapter 3.3.3 --- Ninjurin1 (Ninj1) cDNA transfection in DX5 cells --- p.91 / Chapter 3.3.4 --- mRNA expression of MDR1 in Ninjurin1-transfected DX5 cells (DX5_Ninjl) --- p.93 / Chapter 3.3.5 --- P-glycoprotein expression in Ninjurin1-transfected DX5 cells --- p.95 / Chapter 3.3.6 --- "Cytotoxicity of doxorubicin (Dox) on DX5 control, DX5 vector control and DX5_Ninjl cells" --- p.97 / Chapter 3.3.7 --- "Doxorubicin (Dox) uptake by SA control, DX5 control and DX5_Ninjl cells" --- p.99 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.5 --- Conclusion --- p.105 / Chapter Chapter 4 --- Alternation in MDR1 expression in DX5一THBS1 cells --- p.106 / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials / Chapter 4.2.1.1 --- Cell lines --- p.109 / Chapter 4.2.1.2 --- Cell culture medium； supplements and buffers --- p.109 / Chapter 4.2.1.3 --- Gene expression assay reagents --- p.109 / Chapter 4.2.1.4 --- Western blotting reagents --- p.111 / Chapter 4.2.1.5 --- Plasmid DNA extraction --- p.114 / Chapter 4.2.1.6 --- Transient transfection --- p.115 / Chapter 4.2.1.7 --- MTT reagents --- p.115 / Chapter 4.2.2 --- Methods / Chapter 4.2.2.1 --- Cell culture --- p.117 / Chapter 4.2.2.2 --- Gene expression essay (RT-PCR) --- p.118 / Chapter 4.2.2.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.120 / Chapter 4.2.2.4 --- DNA plasmid extraction --- p.123 / Chapter 4.2.2.5 --- Transient transfection --- p.123 / Chapter 4.2.2.6 --- MTT assay --- p.124 / Chapter 4.2.2.7 --- Quantification of doxorubicin (Dox) uptake by flow cytometry --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1 --- mRNA expression of Thrombospondinl (THBS1) in SA and DX5 cells --- p.126 / Chapter 4.3.2 --- The protein expression of Thrombospondinl (THBS1) in SA and DX5 cells --- p.128 / Chapter 4.3.3 --- Thrombospondinl (THBS1) cDNA transfection in DX5 cells --- p.130 / Chapter 4.3.4 --- mRNA expression of MDR1 in Thrombospondinl-transfected DX5 cells (DX5_THBS1) --- p.132 / Chapter 4.3.5 --- P-glycoprotein expression in Thrombospondinl-transfected DX5 cells --- p.134 / Chapter 4.3.6 --- "Cytotoxicity of doxorubicin (Dox) on DX5 control, DX5 vector control and DX5一THBS1 cells" --- p.136 / Chapter 4.3.7 --- "Doxorubicin (Dox) uptake by SA control, DX5 control and DX5_THBS1 cells" --- p.138 / Chapter 4.4 --- Discussion --- p.141 / Chapter 4.5 --- Conclusion --- p.145 / Chapter Chapter 5 --- General discussion --- p.146 / Chapter 5.1 --- Doxorubicin induced multidrug resistance in human uterin sarcoma cells via upregulation of P-glycoprotein --- p.147 / Chapter 5.2 --- The down-regulation of Ninjurin1 in human uterine sarcoma cells contributed to multidrug resistance --- p.148 / Chapter 5.3 --- The down-regulation of Thrombospondin1 in human uterine sarcoma cells contributed to multidrug resistance --- p.150 / Chapter 5.4 --- Conclusion and Future Perspective --- p.153 / Reference --- p.155

Acute Lymphoblastic Leukemia (ALL) is an aggressive hematologic tumor and is the most common malignancy in children (Horton and Steuber 2014). This disease is characterized by the infiltration of bone marrow by malignant immature lymphoid progenitor cells and is invariably fatal without treatment. Although multi-agent combination chemotherapy is curative in a significant fraction of ALL patients, treatment currently fails in approximately 20% of children and up to 50% of adults with ALL, making relapse and drug resistance the most substantial challenge in the treatment of this disease(Fielding, Richards et al. 2007, Aster and DeAngelo 2013). Understanding what causes treatment failure is of great medical importance as second line therapies also fail in the majority of relapse T-cell ALL (TALL) patients (Fielding, Richards et al. 2007, Aster and DeAngelo 2013). Using next-generation sequencing to compare the genomes of tumors before and after therapy, mutations in gene cytosolic 5’-nucleotidase II (NT5C2) were discovered in 19% of pediatric samples with relapsed T-ALL(Tzoneva, Carpenter et al. 2013). Preliminary structure function analysis and subsequent in vitro experimental nucleotidase activity assays confirmed that these mutations lead to hyperactive NT5C2 protein. Furthermore, NT5C2 mutant proteins conferred resistance to 6-mercaptopurine and 6-thioguanine chemotherapy drugs when expressed in ALL lymphoblasts, suggesting NT5C2 is responsible for the inactivation of nucleoside-analog chemotherapy drugs. In order to assess the ability of these mutations to lead to novel inhibitor schemes, the functional impact of each mutation was analyzed through robust structure function methods. The result of this in silico analysis, is the identification of a potential allosteric regulatory mechanism of negative feedback inhibition never before described. Most notably, the majority of NT5C2 mutations identified have characteristics that suggest they abrogate the function of this proposed mechanism, yielding a novel viable target for the development of allosteric inhibitors specific for constitutively active NT5C2 mutant proteins. Overall these findings support a prominent role for activating mutations in NT5C2 and chemotherapy resistance in ALL, and highlight new avenues for relapsed ALL therapy development in the future.

A terapia fotodinâmica (Photodynamic Therapy - PDT) é um método de tratar neoplasias baseado na interação entre luz, oxigênio molecular e um agente fotossensibilizador. Após a administração do agente, o tumor é iluminado com luz visível, ativando-o e produzindo espécies reativas de oxigênio, altamente citotóxicas, que provocam morte celular e destruição tecidual. Com a destruição do tumor há ativação do sistema imune inato e o subsequente processo inflamatório determina a apresentação de antígenos tumorais aos linfócitos, promovendo uma resposta imunológica adaptativa contra o tecido tumoral. O presente trabalho visou estudar a PDT associando um laser de diodo como fonte de luz e o fotossensibilizante Azul de Metileno (AM) a 1%, avaliando a sua efetividade no tratamento do Tumor de Ehrlich (TE) em sua forma sólida e a resposta imunológica nos animais tratados. Em um primeiro estudo, avaliou-se macro e microscopicamente tumores tratados, determinando a capacidade do protocolo em induzir inflamação e destruição do tecido tumoral. No segundo estudo, a resposta imune foi estudada em camundongos desafiados com um segundo implante de células do tumor de Ehrlich. O primeiro implante tumoral foi tratado com a PDT ou a excisão cirúrgica, comparando-se com um grupo controle sem tratamento. Os parâmetros avaliados após 17 dias foram o crescimento tumoral (p>0,05), peso relativo dos órgãos linfóides [Baço (p<0,05) e Linfonodo poplíteo (p>0,05)], tamanho relativo do linfonodo (p<0,05), presença de metástase em linfonodo poplíteo (p>0,05), contagem de leucócitos sanguíneos (p>0,05) e análise morfométrica quantitativa do tumor secundário [determinação da fração volumétrica de células tumorais (p<0,05), infiltrado inflamatório (p<0,05), necrose (p>0,06) e porcentagem da área tumoral em necrose (p<0,05)]. A PDT com o AM foi capaz de induzir necrose do TE e inflamação, havendo diferenças da resposta imune sistêmica quando comparado aos animais tratados por meio de excisão cirúrgica do tumor de Ehrlich. / Photodynamic therapy (PDT) is a method of treating neoplasms based on the interaction between light, molecular oxygen and a photosensitizing agent. After administration of the photosensitizer, the tumor is illuminated with visible light, activating the agent and producing reactive oxygen species (ROS). This highly cytotoxic ROS cause cell death and tissue destruction. The activation of the innate immune system and the subsequent inflammation induces tumor antigen presentation to lymphocytes, promoting an adaptive immune response against the tumor cells. This work aimed to study the PDT using a diode laser as light source and Methylene Blue (MB) 1% as photosensitizer. It was accessed its effectiveness in treating Ehrlich Solid tumor (ET) and the immune response produced in treated animals. First the treated tumors were evaluated macroscopically and microscopically, determining the ability of the protocol to induce inflammation and tumor tissue destruction. In a second study, the immune response was studied in mice challenged with a second tumor cell implant. The primary tumor was treated with PDT or surgical excision, comparing with a control group without treatment. The parameters evaluated after 17 days were tumor growth (p> 0.05), relative weight of lymphoid organs [spleen (p <0.05) and popliteal lymph node (p> 0.05)], the relative size of the lymph node (p <0, 05), metastasis at lymph node (p>0,05), blood leukocyte count (p> 0.05) and quantitative morphometric analysis of secondary tumor [determining the volume fraction of tumor cells (p <0.05), inflammatory infiltrate (p <0.05), necrosis (p> 0.06) and tumor necrosis area (p <0.05)]. PDT with MB was able to induce necrosis of the ET and inflammation, with differences in the immune response when compared to animals treated surgically to remove the Ehrlich tumor in its solid form.

Yuen, Ka Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 95-104). / Abstracts in English and Chinese. / Chapter 1 --- Introduction / Chapter 1.1 --- CANCER / Chapter 1.1.1 --- OVERVIEW OF CANCER --- p.1 / Chapter 1.1.2 --- DEVELOPMENT OF CANCER --- p.1 / Chapter 1.1.3 --- CHARACTERISTICS OF CANCER CELLS --- p.3 / Chapter 1.1.4 --- CATEGORIZATION OF CANCER --- p.6 / Chapter 1.1.5 --- RISK FACTORS IN CANCER DEVELOPMENT --- p.7 / Chapter 1.1.6 --- EPIDEMIOLOGY OF CANCER --- p.11 / Chapter 1.1.7 --- CANCER THERAPIES --- p.13 / Chapter 1.2 --- SOYBEANS AND ISOFLAVONES / Chapter 1.2.1 --- GENERAL INTRODUCTION OF ISOFLAVONES --- p.18 / Chapter 1.2.2 --- NATURAL FUNCTIONS OF ISOFLAVONES --- p.19 / Chapter 1.2.3 --- STRUCTURES OF ISOFLAVONES --- p.19 / Chapter 1.2.4 --- BIOACTIVITIES OF SOY ISOFLAVONES --- p.20 / Chapter 1.2.5 --- PRODUCTION OF SOY ISOFLAVONES CAN BE AFFECTED BY MANY FACTORS --- p.21 / Chapter 1.3 --- THE AIM AND OBJECTIVES OF THE PROJECT / Chapter 1.3.1 --- AIM OF THE PROJECT --- p.22 / Chapter 1.3.2 --- OBJECTIVES OF THE PROJECT --- p.23 / Chapter 2 --- Materials / Chapter 2.1 --- 19 DROUGHT TOLERANT SOYBEAN LINES --- p.24 / Chapter 2.2 --- 5 HUMAN CANCER CELL LINES --- p.25 / Chapter 2.3 --- CHEMICALS --- p.25 / Chapter 2.4 --- REAGENTS --- p.26 / Chapter 2.5 --- SOLUTIONS --- p.26 / Chapter 2.6 --- MAJOR EQUIPMENTS AND MATERIALS --- p.28 / Chapter 3 --- Methodology / Chapter 3.1 --- PREPARATION OF SOYBEAN EXTRACTS --- p.29 / Chapter 3.2 --- HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC) ANALYSIS OF SOYBEAN EXTRACTS / Chapter 3.2.1 --- PREPARATION OF SOYBEAN EXTRACTS FOR HPLC ANALYS --- p.30 / Chapter 3.2.2 --- HPLC ANALYSIS --- p.30 / Chapter 3.3 --- PREPARATION OF 5 HUMAN CANCER CELL LINES FOR ANTI-PROLIFERATION ASSAY / Chapter 3.3.1 --- THAWING OF THE C Y R O P R E S E R V E D CELL LINES --- p.31 / Chapter 3.3.2 --- MAINTAINING OF CELL LINES --- p.32 / Chapter 3.3.3 --- ANTI-PROLIFERATION TEST WITH MTT ASSAY --- p.33 / Chapter 3.4 --- STATISTICS --- p.35 / Chapter 4 --- Results / Chapter 4.1 --- PREPARARTION OF SOYBEAN EXTRACTS --- p.36 / Chapter 4.2 --- HPLC ANALYSIS OF 5 SELECTED ISOFLAVONES IN 19 SOYBEAN SAMPLES --- p.36 / Chapter 4.3 --- COMPARISON OF SUM OF 5 SELECTED ISOFLAVONES FROM THE PARENT SOYBEAN AND VARIETIES HARVESTED FROM IRRIGATED LAND --- p.43 / Chapter 4.4 --- COMPARISON OF SUM OF 5 SELECTED ISOFLAVONES FROM THE SOYBEAN VARIETIES HARVESTED FROM DROUGHT LAND --- p.45 / Chapter 4.5 --- COMPARISON OF SELECTED ISOFLAVONES FROM THE PARENT SOYBEAN AND VARIETIES HARVESTED FROM IRRIGATED LAND --- p.47 / Chapter 4.6 --- COMPARISON OF SELECTED ISOFLAVONES FROM THE PARENT SOYBEAN AND VARIETIES HARVESTED FROM DAROUGHT LAND --- p.54 / Chapter 4.7 --- COMPARISON OF SUM OF SELECTED ISOFLAVONES AMONG THE SOYBEANS HARVESTED FROM IRRIGATED LAND AND DROUGHT LAND --- p.59 / Chapter 4.8 --- DETERMINATION OF ANTI-PROLIFERATION ABILITIES OF SOYBEAN SAMPLES --- p.19 / Chapter 4.8.1 --- ANTI-PROLIFERATION TEST OF ETHANOL AND 2-PHEN YLCHROMONE --- p.61 / Chapter 4.8.2 --- ANTI-PROLIFERATION ACTIVITIES OF 19 SOYBEAN SAMPLES ON 5 HUMAN CANCER CELL LINES --- p.61 / Chapter 4.9 --- COMPARISON OF ANTI-PROLIFERATION POTENCIES OF19 SOYBEAN SAMPLES WITH SUM OF SELECTED ISOFLAVONES --- p.70 / Chapter 4.10 --- COMPARISON OF ANTI-PROLIFERATION POTENCIES OF19 SOYBEAN SAMPLES --- p.72 / Chapter 4.11 --- ANTI-PROLIFERATION EFFECT OF INDIVIDUAL ISOFLAVONES ON FIVE CANCER CELL LINES --- p.74 / Chapter 5 --- Discussion / Chapter 5.1 --- EXTRACTION OF 19 SOYBEAN LINES --- p.77 / Chapter 5.2 --- DETERMINATION OF QUANTITIES OF SELECTED ISOFLAVONES IN 19 SOYBEAN SAMPLES BY HPLC ANALYSIS --- p.77 / Chapter 5.3 --- COMPARISON OF SELECTED ISOFLAVONES AMONG 19 SOYBEAN SAMPLES / Chapter 5.3.1 --- COMPARISON OF SUM OF SELECTED ISOFLAVONES BETWEEN PARENT AND SOYBEANS HARVESTED FROM IRRIGATED LAND --- p.80 / Chapter 5.3.2 --- COMPARISON OF SUM OF SELECTED ISOFLAVONES BETWEEN SOYBEANS HARVESTED FROM DROUGHT LAND --- p.81 / Chapter 5.3.3 --- COMPARISON OF SELECTED ISOFLAVONES BETWEEN SOYBEANS HARVESTED FROM IRRIGATED LAND --- p.81 / Chapter 5.3.4 --- COMPARISON OF SELECTED ISOFLAVONES BETWEEN SOYBEANS HARVESTED FROM DROUGHT LAND --- p.82 / Chapter 5.3.5 --- COMPARISON OF SUM OF SELECTED ISOFLAVONES BETWEEN SOYBEANS HARVESTED FROM IRRIGATED LAND AND DROUGHT LAND --- p.83 / Chapter 5.4 --- COMPARISON OF ANTI-PROLIFERATION ACTIVITIES OF 19 SOYBEAN SAMPLES / Chapter 5.4.1 --- COMPARISON OF ANTI-PROLIFERATION ACTIVITIES OF19 SOYBEAN SAMPLES AMONG 5 CANCER CELL LINES --- p.84 / Chapter 5.4.2 --- COMPARISON OF ANTI-PROLIFERATION POTENCIES OF19 SOYBEAN SAMPLES --- p.85 / Chapter 5.4.3 --- COMPARISON OF ANTI-PROLIFERATION ACTIVITIES OF19 SOYBEAN SAMPLES AND CORRESPONDING SUM OF SELECTED ISOFLAVONES --- p.86 / Chapter 5.4.4 --- COMPARISON OF IC50S FROM SOYBEANS HARVESTED FROM IRRIGATED LAND AND DROUGHT LAND --- p.87 / Chapter 5.4.5 --- CORRELATION OF ISOFLAVONES AND ANTI-PROLIFERATION POTENCIES --- p.88 / Chapter 6 --- Conclusion --- p.90 / Chapter 7 --- References --- p.91 / Chapter 8 --- Appendix --- p.S1

Lau, Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 223-237). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgments --- p.vi / Publication List --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvi / List of Tables --- p.xx / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.1.1 --- Cancer in Hong Kong --- p.1 / Chapter 1.1.2 --- Different types of cancer treatments and the side effects --- p.4 / Chapter 1.1.3 --- Alternative therapies for cancer treatment --- p.6 / Chapter 1.1.3.1 --- Successful examples of anti-cancer drugs from traditional Chinese herbs --- p.7 / Chapter 1.2 --- Anti-tumor study approaches --- p.11 / Chapter 1.2.1 --- Direct cytotoxic activities --- p.12 / Chapter 1.2.2 --- Immunomodulatory activities --- p.14 / Chapter 1.2.3 --- Anti-angiogenesis activities --- p.16 / Chapter 1.3 --- Objectives of our study --- p.20 / Chapter Chapter 2 --- Background of selected Chinese herbs in our study / Chapter 2.1 --- Search for anti-tumor Chinese herbs --- p.21 / Chapter 2.1.1 --- Chinese herbs commonly used for cancer treatment --- p.21 / Chapter 2.1.2 --- Literature Search --- p.21 / Chapter 2.2 --- Results --- p.22 / Chapter 2.2.1 --- Lists of Chinese herbs from various Chinese medicine practitioners --- p.22 / Chapter 2.2.2 --- Selected traditional Chinese herbs from literature search --- p.22 / Chapter 2.2.3 --- Selected Chinese herbs for our study --- p.27 / Chapter 2.3 --- Background information of the five selected Chinese herbs --- p.28 / Chapter 2.3.1 --- Fructus Bruceae (FB) --- p.28 / Chapter 2.3.1.1 --- Traditional uses --- p.28 / Chapter 2.3.1.2 --- Previous Studies of Fructus Bruceae --- p.28 / Chapter 2.3.1.3 --- Isolated compounds of FB --- p.31 / Chapter 2.3.2 --- Cortex Phellodendri Amurensis (PA) --- p.35 / Chapter 2.3.2.1 --- Traditional uses --- p.35 / Chapter 2.3.2.2 --- Previous studies of Cortex Phellodendri Amurensis --- p.35 / Chapter 2.3.2.3 --- Previous studies of Berberine --- p.38 / Chapter 2.3.3 --- Radix et Rhizoma Asteris (RA) --- p.39 / Chapter 2.3.3.1 --- Traditional uses --- p.39 / Chapter 2.3.3.2 --- Previous Studies of Radix et Rhizoma Asteris --- p.39 / Chapter 2.3.4 --- Semen Coicis (SC) --- p.41 / Chapter 2.3.4.1 --- Traditional uses --- p.41 / Chapter 2.3.4.2 --- Previous Studies of Semen Coicis --- p.41 / Chapter 2.3.5 --- Radix Scrophulariae (RS) --- p.43 / Chapter 2.3.5.1 --- Traditional uses --- p.43 / Chapter 2.3.5.2 --- Previous Studies of Radix Scrophulariae --- p.43 / Chapter 2.4 --- Authentication of selected Chinese herbs --- p.45 / Chapter 2.4.1 --- Sources --- p.45 / Chapter 2.4.2 --- Morphological characteristics of the Chinese herbs --- p.47 / Chapter 2.4.2.1 --- Fructus Bruceae --- p.47 / Chapter 2.4.2.2 --- Cortex Phellodendri Amurensis --- p.48 / Chapter 2.4.2.3 --- Radix et Rhizoma Asteris --- p.49 / Chapter 2.4.2.4 --- Semen Coicis --- p.50 / Chapter 2.4.2.5 --- Radix Scrophulariae --- p.51 / Chapter 2.5 --- Extraction of selected Chinese herbs --- p.52 / Chapter 2.5.1 --- Materials and methods --- p.52 / Chapter 2.5.1.1 --- Preparation of aqueous extracts of selected Chinese herbs --- p.52 / Chapter 2.5.2 --- Results --- p.53 / Chapter 2.5.2.1 --- Percentage yield of aqueous extract of selected Chinese herbs --- p.53 / Chapter 2.6 --- Discussion --- p.54 / Chapter Chapter 3 --- Direct cytotoxic effect of selected Chinese herbs / Chapter 3.1 --- Background --- p.55 / Chapter 3.2 --- Materials and methods --- p.56 / Chapter 3.2.1 --- Cell cultures --- p.56 / Chapter 3.2.2 --- Determination of cell viability by MTT assay --- p.58 / Chapter 3.2.3 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.59 / Chapter 3.2.4 --- Preparation of etoposide for direct cytotoxic assay --- p.60 / Chapter 3.2.5 --- Statistical analysis --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Cytotoxic effects of five selected Chinese herbs on a panel of human cancer cell lines and human normal cell line --- p.62 / Chapter 3.3.2 --- Comparison of the cytotoxic effect of etoposide and the selected Chinese herbal extracts on a panel of human tumor cells --- p.72 / Chapter 3.3.3 --- Further investigations of the anti-tumor effect of PA --- p.75 / Chapter 3.3.3.1 --- Materials and methods --- p.75 / Chapter 3.3.3.1.1 --- Quantification of berberine chloride in PA aqueous extract using TLC --- p.75 / Chapter 3.3.3.1.2 --- Determination of cell viability by MTT assay --- p.76 / Chapter 3.3.3.2 --- Results --- p.76 / Chapter 3.3.3.2.1 --- Quantification of berberine chloride in PA aqueous extract using TLC --- p.76 / Chapter 3.3.3.2.2 --- Cytotoxic effect of berberine on a panel of human cancer cell lines --- p.78 / Chapter 3.4 --- Discussion --- p.80 / Chapter Chapter 4 --- Immunomodulatory effects of selected Chinese herbs / Chapter 4.1 --- Background --- p.84 / Chapter 4.2 --- Materials and methods --- p.87 / Chapter 4.2.1 --- Preparation of human peripheral blood mononuclear cells (huPBMCs) --- p.87 / Chapter 4.2.2 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.88 / Chapter 4.2.3 --- Preparation of cell mitogens --- p.88 / Chapter 4.2.4 --- Statistical analysis --- p.89 / Chapter 4.3 --- Results --- p.89 / Chapter 4.3.1 --- Mitogenic activities of the selected herbal extracts on huPBMCs --- p.89 / Chapter 4.4 --- Further investigations of the mitogenic activities of SC and RA extracts --- p.96 / Chapter 4.4.1 --- Materials and methods --- p.96 / Chapter 4.4.1.1 --- Preparation of human peripheral blood mononuclear cells (huPBMCs) --- p.96 / Chapter 4.4.1.2 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.96 / Chapter 4.4.2 --- Results --- p.97 / Chapter 4.4.2.1 --- Mitogenic effects of SC and RA aqueous extracts (in the presence of polymyxin B) --- p.97 / Chapter 4.5 --- Chemical characterization of RA aqueous extract --- p.100 / Chapter 4.5.1 --- Materials and methods --- p.100 / Chapter 4.5.1.1 --- Quantification of polysaccharide and carbohydrate contents in RA aqueous extract --- p.100 / Chapter 4.5.1.2 --- Quantification of protein content in RA aqueous extract --- p.101 / Chapter 4.5.2 --- Results --- p.103 / Chapter 4.5.2.1 --- Chemical characterization of RA aqueous extract --- p.103 / Chapter 4.6 --- Further investigations of the underlying mechanisms of the mitogenic activities of RA aqueous extract --- p.104 / Chapter 4.6.1 --- Materials and methods --- p.104 / Chapter 4.6.1.1 --- Preparation of human peripheral blood mononuclear cells (huPBMCs) --- p.104 / Chapter 4.6.1.2 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.104 / Chapter 4.6.1.3 --- Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) --- p.105 / Chapter 4.6.1.4 --- Statistical analysis --- p.106 / Chapter 4.6.2 --- Results --- p.106 / Chapter 4.6.2.1 --- Effects of RA aqueous extract on productions of cytokinesin huPBMCs --- p.106 / Chapter 4.7 --- Discussion --- p.108 / Chapter Chapter 5 --- Anti-angiogenesis effects of selected Chinese herbs / Chapter 5.1 --- Background of in vivo zebrafish model --- p.112 / Chapter 5.2 --- Materials and methods --- p.117 / Chapter 5.2.1 --- Maintenance of zebrafish --- p.117 / Chapter 5.2.2 --- Collection of zebrafish embryos --- p.117 / Chapter 5.2.3 --- Zebrafish embryos treated with different herbal extracts --- p.117 / Chapter 5.2.4 --- Visual screens of zebrafish embryos using fluorescence microscopy --- p.118 / Chapter 5.2.5 --- Statistical analysis --- p.118 / Chapter 5.3 --- Results --- p.120 / Chapter 5.3.1 --- Anti-angiogenesis effect of SU5416 --- p.120 / Chapter 5.3.2 --- Anti-angiogenesis effects of selected herbal extracts on zebrafish model --- p.122 / Chapter 5.4 --- Discussion --- p.133 / Chapter Chapter 6 --- Further investigations on the anti-tumor effects of Fructus Bruceae and its sub-fractions / Chapter 6.1 --- Introduction --- p.136 / Chapter 6.2 --- Solvent partition of FB aqueous extract --- p.138 / Chapter 6.2.1 --- Materials and methods --- p.138 / Chapter 6.2.1.1 --- Solvent partition --- p.138 / Chapter 6.2.1.2 --- Thin layer chromatography of FB fractions --- p.138 / Chapter 6.2.2 --- Results --- p.139 / Chapter 6.2.2.1 --- Percentage yield of different fractions of FB aqueous extract --- p.139 / Chapter 6.2.2.2 --- Thin layer chromatography of FB fractions --- p.140 / Chapter 6.3 --- Investigations of the anti-tumor activities of FBW fraction --- p.141 / Chapter 6.3.1 --- Materials and methods --- p.141 / Chapter 6.3.1.1 --- Cell cultures --- p.141 / Chapter 6.3.1.2 --- Determination of cell viability by MTT assay --- p.141 / Chapter 6.3.1.3 --- Preparation of human peripheral blood mononuclear cells (huPBMCs) --- p.141 / Chapter 6.3.1.4 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.141 / Chapter 6.3.1.5 --- Statistical analysis --- p.141 / Chapter 6.3.2 --- Results --- p.142 / Chapter 6.3.2.1 --- Cytotoxic effects of FBW on a panel of human cancer cells and human normal cells --- p.142 / Chapter 6.3.2.2 --- Mitogenic activities of FBW fraction on huPBMCs --- p.145 / Chapter 6.4 --- Chemical characterizations of FB aqueous extract and FBW fraction --- p.147 / Chapter 6.4.1 --- Materials and methods --- p.147 / Chapter 6.4.2 --- Results --- p.147 / Chapter 6.5 --- Bioassay guided fractionation of FBW --- p.149 / Chapter 6.5.1 --- Fractionation using macroporous resin column (D101) --- p.149 / Chapter 6.5.2 --- Investigations of the anti-tumor effects of the sub-fractions of FBW --- p.151 / Chapter 6.5.2.1 --- Direct cytotoxic effects of FBW sub-fractions on NB-4 cells and human normal cells --- p.151 / Chapter 6.5.2.2 --- Immunomodulatory effects of FBW-DH sub-fraction --- p.154 / Chapter 6.5.3 --- Fractionation using ethanol precipitation --- p.155 / Chapter 6.5.3.1 --- Chemical characterization of sub-fractions of FBW-DH --- p.156 / Chapter 6.5.3.2 --- "Direct cytotoxic effects of 50P, 80P and 80S on NB-4 cells and human normal cells" --- p.159 / Chapter 6.5.3.2.1 --- DNA agarose gel electrophoresis --- p.163 / Chapter 6.5.3.2.2 --- Cell death detection ELISA --- p.166 / Chapter 6.5.3.2.3 --- ELISA of apoptotic related proteins --- p.168 / Chapter 6.5.3.2.4 --- Telomerase PCR ELISA --- p.176 / Chapter 6.5.3.3 --- "Immunomodulatory effects of 50P, 80P and 80S" --- p.178 / Chapter 6.5.3.3.1 --- Human Thl/Th2 cytokine cytometric bead array (CBA) --- p.180 / Chapter 6.5.3.3.2 --- Limulus Amebocyte Lysate assay --- p.183 / Chapter 6.5.3.4 --- "Anti-angiogenic effects of 50P, 80P and 80S" --- p.184 / Chapter 6.5.3.5 --- Liquid chromatography mass spectrometry (LCMS) analysis of 50P --- p.192 / Chapter 6.6 --- Discussion --- p.194 / Chapter Chapter 7 --- General discussions and conclusions / Chapter 7.1 --- Anti-tumor activities of five selected Chinese herbs --- p.202 / Chapter 7.2 --- Significance of the present study --- p.213 / Chapter 7.3 --- Limitations of our study --- p.214 / Chapter 7.4 --- Future work --- p.215 / Appendices / Appendix I Phenol-sulphuric acid spectrophotometric assay --- p.216 / Appendix II Bradford assay --- p.217 / Appendix III Calibration curves of cytokines in CBA assay --- p.218 / Appendix IV Endotoxin standard curve --- p.220 / Appendix V LCMS data of two chemical markers of FB --- p.221 / Bibliography --- p.223

A terapia fotodinâmica (Photodynamic Therapy - PDT) é um método de tratar neoplasias baseado na interação entre luz, oxigênio molecular e um agente fotossensibilizador. Após a administração do agente, o tumor é iluminado com luz visível, ativando-o e produzindo espécies reativas de oxigênio, altamente citotóxicas, que provocam morte celular e destruição tecidual. Com a destruição do tumor há ativação do sistema imune inato e o subsequente processo inflamatório determina a apresentação de antígenos tumorais aos linfócitos, promovendo uma resposta imunológica adaptativa contra o tecido tumoral. O presente trabalho visou estudar a PDT associando um laser de diodo como fonte de luz e o fotossensibilizante Azul de Metileno (AM) a 1%, avaliando a sua efetividade no tratamento do Tumor de Ehrlich (TE) em sua forma sólida e a resposta imunológica nos animais tratados. Em um primeiro estudo, avaliou-se macro e microscopicamente tumores tratados, determinando a capacidade do protocolo em induzir inflamação e destruição do tecido tumoral. No segundo estudo, a resposta imune foi estudada em camundongos desafiados com um segundo implante de células do tumor de Ehrlich. O primeiro implante tumoral foi tratado com a PDT ou a excisão cirúrgica, comparando-se com um grupo controle sem tratamento. Os parâmetros avaliados após 17 dias foram o crescimento tumoral (p>0,05), peso relativo dos órgãos linfóides [Baço (p<0,05) e Linfonodo poplíteo (p>0,05)], tamanho relativo do linfonodo (p<0,05), presença de metástase em linfonodo poplíteo (p>0,05), contagem de leucócitos sanguíneos (p>0,05) e análise morfométrica quantitativa do tumor secundário [determinação da fração volumétrica de células tumorais (p<0,05), infiltrado inflamatório (p<0,05), necrose (p>0,06) e porcentagem da área tumoral em necrose (p<0,05)]. A PDT com o AM foi capaz de induzir necrose do TE e inflamação, havendo diferenças da resposta imune sistêmica quando comparado aos animais tratados por meio de excisão cirúrgica do tumor de Ehrlich. / Photodynamic therapy (PDT) is a method of treating neoplasms based on the interaction between light, molecular oxygen and a photosensitizing agent. After administration of the photosensitizer, the tumor is illuminated with visible light, activating the agent and producing reactive oxygen species (ROS). This highly cytotoxic ROS cause cell death and tissue destruction. The activation of the innate immune system and the subsequent inflammation induces tumor antigen presentation to lymphocytes, promoting an adaptive immune response against the tumor cells. This work aimed to study the PDT using a diode laser as light source and Methylene Blue (MB) 1% as photosensitizer. It was accessed its effectiveness in treating Ehrlich Solid tumor (ET) and the immune response produced in treated animals. First the treated tumors were evaluated macroscopically and microscopically, determining the ability of the protocol to induce inflammation and tumor tissue destruction. In a second study, the immune response was studied in mice challenged with a second tumor cell implant. The primary tumor was treated with PDT or surgical excision, comparing with a control group without treatment. The parameters evaluated after 17 days were tumor growth (p> 0.05), relative weight of lymphoid organs [spleen (p <0.05) and popliteal lymph node (p> 0.05)], the relative size of the lymph node (p <0, 05), metastasis at lymph node (p>0,05), blood leukocyte count (p> 0.05) and quantitative morphometric analysis of secondary tumor [determining the volume fraction of tumor cells (p <0.05), inflammatory infiltrate (p <0.05), necrosis (p> 0.06) and tumor necrosis area (p <0.05)]. PDT with MB was able to induce necrosis of the ET and inflammation, with differences in the immune response when compared to animals treated surgically to remove the Ehrlich tumor in its solid form.

Gold nanoparticle has emerged as an attractive platform for drug delivery applications by complementing the existing drug delivery carriers. Currently, only a few gold nanoparticle-based anticancer drug delivery systems have been reported, compared to the polymer-based delivery systems. Additionally, there is still a lack of understanding for the behavior and fate of the gold-drug conjugate in the body that further attention is required. The overall goal of this thesis is to investigate the in vivo behavior of colloidal gold nanoparticle and its therapeutic efficacy in an animal model, especially in a drug delivery application. To achieve this goal, we investigated the feasibility of using colloidal gold nanoparticle as an anticancer agent delivery vehicle for treatment of cancer. Then, long-term clearance, toxicity, and biodistribution of colloidal gold nanoparticle were studied to further aid in understanding of using colloidal gold nanoparticle as a drug delivery platform. In particular, two representative sizes of gold nanoparticles, 5nm and 60nm, were investigated for the size effect on the therapeutic efficacy, toxicity, biodistribution, and clearance in cancer nanotherapy. We believe that nanoparticle size plays a critical role in not only delivering the drug to the target site but also determining the in vivo behavior such as biodistribution and clearance. By choosing an appropriate size scale for the system, we successfully used the small-sized gold nanoparticles for drug delivery applications, which also displayed no apparent toxicity with desirable clearance from the biological system. This work is significant by providing an insight on a potential ideal candidate for drug delivery carrier for cancer nanotherapy.

Histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated a wealth of biological effects, including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. The recent FDA approvals of the inhibitors SAHA and FK-228 have validated HDACi clinical use in cutaneous T cell lymphoma, while numerous clinical trials are currently ongoing using HDACi against a variety of disease states. While the future of the HDAC field looks increasingly promising, there are lingering issues hindering broader use. Recent data point to dysregulation of specific HDAC isoforms in many disease states. However, most current HDACi are pan-inhibitors, lacking the specificity to target individual isoforms. Adding to this, there are currently 18 identified HDAC isoforms, and most lack a defined crystal structure, further complicating the task of designing isoform-specific inhibitors. Most importantly, HDACi have demonstrated a lack of efficacy against solid tumors in the clinic, a major obstacle to broader use in cancer therapy. Several of these issues could more fully be addressed through specific targeting of HDACi, and could bring HDACi into wider and more efficacious pharmaceutical use. Targeting the specific tissue or organelle where HDAC dysregulation occurs could confer greater efficacy in vivo. To this end, we have created four classes of compounds: (1) aryltriazolyl HDACi that potently inhibit HDAC activity and prostate cancer cell growth, (2) dual-targeted inhibitors of Topoisomerase II and HDAC and (3) dual-targeted inhibitors of Topoisomerase I and HDAC, both of which have potent inhibition against both target enzymes as well as cancer cell lines, and finally (4) macrocyclic HDACi that potently inhibit the growth of lung cancer cell lines and preferentially target lung tissue in vivo.

published_or_final_version / Public Health / Master / Master of Public Health