published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

 Ischemic stroke is the leading cause of death and disability in human diseases all around the world. As effective treatment for ischemic stroke is still absent, seeking for new therapy is of great interest. Currently, several key pathological cascades following cerebral ischemia have been explored to develop further therapies. Among them, reactive nitrogen species (RNS) has been indicated to play a critical role in cerebral ischemia reperfusion injury. As one of the RNS, peroxynitrite contributes to the neural cell death and subsequent brain dysfunction in the process. Thus, development of antioxidants targeting on peroxynitrite could be an important strategy for the treatment of cerebral ischemia-reperfusion injury. Baicalin is a polyphenolic compound isolated from roots of Scutellaria baicalensis. Baicalin exerted protective effects against cerebral ischemia-reperfusion injury but the mechanisms are not clear yet. In this study, we investigated the free radical scavenging ability and neuroprotective effects of baicalin. According to our results, baicalin neutralized DPPH radicals effectively. By using electron paramagnetic resonance (EPR) spin trapping technology and fluorescent probe DAF-2DA, we found that baicalin dose-dependently scavenged superoxide, but had very low effect on elimination of nitric oxide. The immunofluoresent results revealed that baicalin at the concentration of 50 M completely suppressed the nitrotyrosine formation induced by 3-morpholinylsydnoneimine chloride (SIN-1, a peroxynitrite donor) in neuroblastoma SH-SY5Y cells. Mass spetrum provided direct evidence of the peroxynitrite scavenging ability of baicalin. Using MTT assays, we found that baicalin totally reversed peroxynitrite-induced cytotoxicity in SH-SY5Y cells and protected SH-SY5Y cells in oxygen glucose deprivation (OGD) and following reoxygenation injury. Furthermore, in vivo experiments revealed that intravenous injection of baicalin exerted better neuroprotective effect than intraperitoneal administration in rats underwent middle cerebral artery occlusion (MCAO). After cerebral ischemia reperfusion, rats treated with 3 mg/kg of peroxynitrite decomposition catalyst (FeTMPyP) or 25 mg/kg of baicalin revealed a smaller size of infarction volume, suppressed neural cell death and reduced nitrotyrosine formation than MCAO rats. However, baicalin did not alter the expression of tight junction proteins, claudin-5 and ZO-1, in brain endothelial bEnd3 cell line treated with OGD following reoxygenation. In cerebral ischemia reperfusion rats, administration of FeTMPyP at the dosage of 3 mg/kg diminished the Evans blue leakage caused by blood brain barrier disruption, whereas treatment of baicalin did not show significant effect. In conclusion, this study suggests that baicalin can scavenge peroxynitrite and protect neural cells from peroxynitrite-induced injury. Furthermore, baicalin could prevent brains from cerebral ischemia-reperfusion injury and the neuroprotective mechanisms are associated with the scavenging effects on peroxynitrite. These findings provide new insights into the antioxidant and neuroprotective properties of baicalin and indicate the potential application of baicalin for the treatment of ischemic stroke. / published_or_final_version / Chinese Medicine / Master / Master of Philosophy

According to the results, Pa-PDT showed inhibitory effect on MDA-MB-231 cells in vitro with an IC50 value of 0.5 muM at 24 h. Pa-PDT was demonstrated to activate intracellular mitogen activated protein kinases (MAPK) pathways via reactive oxygen species (ROS) production. Pa-PDT IS also believed to induce extracellular signal-regulated kinase (ERK)-mediated autophagy and endoplasmic reticulum stress. Pa-PDT in combination with Tamoxifen is demonstrated to exert a synergetic effect in inhibiting cancer growth. The combination treatment induces both intrinsic and extrinsic apoptosis. Regarding the direct cancer cell killing activity, two dimensional gel electrophoresis screening revealed that Pa-PDT regulates proteins which involve in human leukocyte antigen (HLA) class I-restricted antigen-processing machinery. This activation of antigen presentation was confirmed by Western blot analysis and immunostaining. Furthermore, a cross-presentation of antigen with HLA class I proteins and 70-kDa heat shock protein was found in Pa-PDT-treated cells, as shown by the fluorescent microscopic observation and immunoprecipitation assay. Moreover, the immunogenicity of breast cancer cells was increased by Pa-PDT treatment that triggered phagocytic activity by human macrophages. Our findings provide the first evidence that Pa-PDT can trigger both apoptosis and anti-tumour immunity. / Cancer is one of the most lethal diseases worldwide. Treatments of cancer comprise surgical intervention, radiotherapy or chemotherapy; however, their side effects are still need to be overcome. In order to search for anti-cancer treatments with milder side effects and higher efficiency, traditional Chinese medicine (TCM) has been investigated. Previous study in our laboratory reported that pheophorbide a (Pa), an active compound purified from Scutellaria barbata, combined with photodynamic therapy (PDT) approach produces anti-tumour effect in a wide range of human cancers. Because of the lack of protocols for curing late phase breast cancer, my project is to investigate the therapeutic potential of Pa-PDT and its action mechanism on human breast cancer. A human breast cancer cell line MDA-MB-231, which is estrogen receptor nude and resistant to a conventional breast cancer drug tamoxifen, was used as an in vitro tumour model in my study to mimic the late stage of breast cancer. / Pheophorbide a (Pa) has been proposed to be a potential photosensitizer for the photodynamic therapy of human cancer. However, the immunomodulatory effect of Pa, in the absence of irradiation, has not yet been investigated. The present study revealed that Pa possessed immunostimulating effect on a murine macrophages cell line RAW 264.7. Pa could stimulate the growth of RAW 264.7 cells with the maximal effect at 0.5 muM after 48 h of treatment, where MAPK family including c-Jun N-tenninal kinase (JNK), ERK and p38 MAPK were activated by Pa treatment in a dose-dependent manner. Moreover, the induction of interleukin-6 and tumour necrosis factor-a secretion, and the enhancement of phagocytic activity were observed in Pa-treated RAW 264.7 cells. The results were similar in Pa-treated human immune competent cells (e.g. CD4+ and CD14+ cells) at higher Pa concentrations (from 1 to 10 muM). The present work is the first report to demonstrate the potential immunomodulatory effects of Pa on immune competent cells, apart from its well-known anti-tumour activity. / Bui Xuan, Ngoc Ha. / "December 2010." / Advisers: Fung Kwok Pui; Wong Chun Kwok. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-144). / 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.

Hoi, Wan Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 212-245). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cheung, Tsz Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 97-116). / Abstracts in English and Chinese. / Thesis/Assessment Committee Members --- p.ii / Declaration for Plagiarism and Copyright --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Abbreviations --- p.xii / List of Figures --- p.xv / List of Tables --- p.xvi / Chapter CHAPTER TWO: --- Introduction / Chapter 1.1 --- Background Information / Chapter 1.1.1 --- Breast Cancer --- p.1 / Chapter 1.1.2 --- General Statistics of Breast Cancer Worldwide and in Hong Kong --- p.1 / Chapter 1.1.3 --- Risk Factors for Breast Cancer --- p.2 / Chapter 1.1.4 --- Breast Cancer Treatment and Side Effects --- p.2 / Chapter 1.1.5 --- Types of Breast Cancer --- p.3 / Chapter 1.2 --- Estrogen and Estrogen Receptor / Chapter 1.2.1 --- Estrogen --- p.4 / Chapter 1.2.2 --- Estrogen Receptor --- p.5 / Chapter 1.2.3 --- Estrogen Receptor mediated Gene Transcription --- p.5 / Chapter 1.2.4 --- Estrogen Receptor Alpha and Estrogen Receptor Beta --- p.6 / Chapter 1.2.5 --- Estrogen Receptor Positive Breast Cancer and Treatment --- p.7 / Chapter 1.3 --- Estrogen metabolism and Cytochrome P450 family 1 (CYP1) members / Chapter 1.3.1 --- Estrogen Metabolism in Human --- p.9 / Chapter 1.3.2 --- CYP1A1 and CYP1B1 --- p.9 / Chapter 1.3.3 --- Estrogen Metabolism in Breast --- p.10 / Chapter 1.3.4 --- Carcinogenesis of Estrogens and Estrogen Metabolites --- p.13 / Chapter 1.3.5 --- The Importance of CYP1B1 in Carcinogenesis --- p.15 / Chapter 1.4 --- Aryl Hydrocarbon Receptor / Chapter 1.4.1 --- General Information of Aryl Hydrocarbon Receptor --- p.16 / Chapter 1.4.2 --- Signaling/Regulation Pathways of Aryl Hydrocarbon Receptor --- p.17 / Chapter 1.4.3 --- Crosstalk with Estrogen Receptor --- p.17 / Chapter 1.5 --- Introduction of Herba Scutellaria Barbata and its active ingredient Pheophorbide a --- p.19 / Chapter 1.6 --- Hyposthesis and Objectives --- p.21 / Chapter CHAPTER TWO: --- Direct Cytotoxic/Cytostatic Effect of Pheophorbide a / Chapter 2.1 --- Backgrounds --- p.22 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Chemicals --- p.24 / Chapter 2.2.2 --- Cell Lines --- p.26 / Chapter 2.2.3 --- "Cell Culture Mediums, Buffers and Consumables" / Chapter 2.2.3.1 --- Roswell Park Memorial Institute Tissue Culture Medium1640 (RPMI1640) --- p.26 / Chapter 2.2.3.2 --- RPMI 1640 (Phenol Red-free) --- p.26 / Chapter 2.2.3.3 --- Serum supplement - Fetal Bovine Serum (FBS) --- p.27 / Chapter 2.2.3.4 --- Serum supplement - Charcoal/Dextran Stripped FBS --- p.27 / Chapter 2.2.3.5 --- Antibiotics - Penicillin-Streptomycin (P/S) --- p.27 / Chapter 2.2.3.6 --- Trypsin (0.25%) with EDTA --- p.27 / Chapter 2.2.3.7 --- Trypsin (2.5%) (Phenol Red-free) with EDTA --- p.28 / Chapter 2.2.3.8 --- Dulbeccóةs Phosphate-Buffered Saline (D-PBS) --- p.28 / Chapter 2.2.3.9 --- Tissue Culture Flasks and Multi-well Plate --- p.28 / Chapter 2.2.3.10 --- Trypan Blue Solution --- p.29 / Chapter 2.2.4 --- Reagents for Direct Cytotoxity Test / Chapter 2.2.4.1 --- MTT Assay --- p.29 / Chapter 2.2.4.2 --- Tritiated Thymidine Incorporation Assay --- p.29 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Cell Culture --- p.30 / Chapter 2.3.2 --- Direct Cytotoxicity/Cytostatic Test / Chapter 2.3.2.1 --- MTT Assay --- p.31 / Chapter 2.3.2.2 --- Tritiated Thymidine Incorporation Assay --- p.32 / Chapter 2.3.3 --- Statistical Analysis --- p.32 / Chapter 2.4 --- Results / Chapter 2.4.1 --- The Cytotoxic Effect of Pheophorbide a --- p.34 / Chapter 2.4.2 --- The Combine Effect of Pheophorbide a with 17-β Estradiol and Tamoxifen Citrate --- p.34 / Chapter 2.5 --- Discussions --- p.48 / Chapter CHAPTER THREE: --- Mechanistic Study of Pheophorbide a / Chapter 3.1 --- Backgrounds --- p.53 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Real time PCR / Chapter 3.2.1.1 --- General Chemicals and Equipments --- p.54 / Chapter 3.2.1.2 --- RNA isolation --- p.55 / Chapter 3.2.1.3 --- Reverse Transcription --- p.55 / Chapter 3.2.1.4 --- Real Time PCR --- p.56 / Chapter 3.2.2 --- Western Blotting / Chapter 3.2.2.1 --- Microsome Isolation --- p.58 / Chapter 3.2.2.2 --- Measurement of Protein Concentration --- p.58 / Chapter 3.2.2.3 --- Western Blotting --- p.58 / Chapter 3.2.3 --- Estrogen Metabolism Assay / Chapter 3.2.3.1 --- Chemicals --- p.59 / Chapter 3.2.3.2 --- Estrogen Metabolites Extraction --- p.60 / Chapter 3.2.3.3 --- Liquid Chromatography/Mass Spectrometry --- p.60 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Real time PCR / Chapter 3.3.1.1 --- Cell Culture --- p.61 / Chapter 3.3.1.2 --- RNA Isolation and Reverse Transcription --- p.61 / Chapter 3.3.1.3 --- Real Time PCR --- p.62 / Chapter 3.3.2 --- Western Blotting / Chapter 3.3.2.1 --- Cell Culture --- p.63 / Chapter 3.3.2.2 --- Microsome Isolation --- p.63 / Chapter 3.3.2.3 --- Measurement of Protein Concentration --- p.64 / Chapter 3.3.2.4 --- Western Blotting --- p.64 / Chapter 3.3.3 --- Estrogen Metabolism Assay / Chapter 3.3.3.1 --- Preparation of Calibration Standard --- p.65 / Chapter 3.3.3.2 --- Cell Culture --- p.66 / Chapter 3.3.3.3 --- Estrogen Metabolites Extraction --- p.66 / Chapter 3.3.3.4 --- Liquid Chromatography/Mass Spectrometry --- p.67 / Chapter 3.3.4 --- Statistical Analysis --- p.68 / Chapter 3.4 --- Results --- p.69 / Chapter 3.5 --- Discussions --- p.80 / Chapter CHAPTER FOUR: --- Overall Conclusion and Future Directions / Chapter 4.1 --- Significance of the Study --- p.87 / Chapter 4.2 --- Overall Conclusion --- p.87 / Chapter 4.3 --- Limitation and Difficulties of the Study --- p.89 / Chapter 4.4 --- Future Directions --- p.89 / Appendices / "Appendix I The Melting Curve of real time PCR for β-actin, CYP1A1 and CYP1B1" --- p.92 / Appendix II The Calibration Curve of BSA for Protein Concentration Measurement --- p.93 / Appendix III The Representative Peak of Estradiol Metabolite Standards with corresponding Retention Time --- p.94 / Appendix IV The Calibration Curve of Different Estrogen Metabolites for LC/MS --- p.95 / Appendix V The Accuracy and Precision of Quality Control of Estradiol Metabolites --- p.96 / Bibliography --- p.97

Both mRNA and protein expression levels of P-glycoprotein, one of the major factors involved in drug resistance, was decreased in Pa-treated R-HepG2 cells. The chemo-sensitivity of these MDR cells towards doxorubicin would be enhanced by pretreatment of Pa. / In the study, 35 TCMs with historical background in treating liver diseases were screened. S. barbata was chosen for intensive studies based on its significant anti-hepatoma activity. Using bioassay-guided purification approach, an active component, pheophorbide a (Pa) - a chlorophyll derivative, was isolated from Scutellaria barbata. / Motivated by the severe health hazards worldwide caused by liver cancer, and the pronounced side effects of some recent anti-hepatoma agents in clinical treatment, we have initiated a research project in screening safe and effective agents from Traditional Chinese Medicine (TCM) for the treatment of hepatoma. The main objective of this research is to define the in vitro and in vivo anti-proliferative activities and to identify the action mechanisms of a TCM, the aerial part of Scutellaria barbata , in human hepatoma cells (HepG2 and Hep3B cells). / Pa exhibited anti-proliferative effects on HepG2 and Hep3B cells, through cell-cycle arrest and apoptosis, with IC50 values being 12.5 and 25.7 muM respectively. However, Pa produced insignificant cytotoxic effect on WRL-68 cells, a normal hepatic cell line. Pa also caused cell death in R-HepG2 cells, a multi-drug resistant (MDR) cell line developed from HepG2 cells. Microarray analysis indicated that a hypothetical protein FLJ10803 was found to be down-regulated upon the treatment of Pa on HepG2 cells. The sub-cellular localization of FLJ10803 was demonstrated by over-expression of the GFP fusion protein in HepG2 cells. / The anti-tumor effects of Pa could be enhanced by photodynamic therapy (PDT) approach, presumably due to the rapid generation of reactive oxygen species in the drug-binding site. Pa-PDT showed potent cytotoxicity on hepatoma cell lines, HepG2 and Hep3B, with IC50 values being 0.4 and 1.5 muM, respectively. The antitumor effects were confirmed by studies using animal model, where Pa treatment (300mug/kg/day, s.c.) could significantly inhibit the growth of Hep3B cells in nude mice after PDT treatment in vivo. Fluorescent imaging showed that Pa was located at the mitochondria, and the induction of cell death was found to be initiated by the mitochondrial dependent apoptotic pathway. Results of 2D-gel analysis suggested that Pa-PDT activated an immune-marker expression pathway that results in an over expression of HLA class I proteinsin Pa-PDT treated HepG2 cells. / To conclude, Pa may be a candidate for further development into an anti-hepatomic agent for clinical application. / Tang, Ming Kuen. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4742. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 227-243). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.