A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Masters of Science in Medicine (Pharmacology) Johannesburg, 2013 / Colon cancer is the third most common cancer worldwide and the second most common in the western world. More than 40 % of colon cancer sufferers develop metastases and chemotherapy is often used alone or in combination with radiotherapy as adjunctive therapy for the advanced disease. A major effort has been made in the past decade to develop anticancer agents through both empiric screening and rational design of new compounds. These attempts are made to improve the survival rate, reduce the severe adverse effects associated with existing cancer chemotherapeutic agents as well as to reduce the development of drug resistance. In the present study, two colon cancer cell lines were exposed to novel imidazo[1,2-a]pyridines and novel nucleoside analogues, aiming to investigate the cytotoxic efficacy on the cells, the mode of cell death, and to explore the pathways by which cell death was induced.

Colorectal cancer has a 9.8% cumulative incidence rate, making it the third most common cancer in the Western world. Despite a 50-60% response rate in patients to current cancer therapies, drug resistance and tumour relapse remain a concern. While current therapies reduce the tumour mass, they possibly fail to eradicate a unique population of pluripotent tumour resident cells. These cells, known as cancer stem cells, may have similar properties of self-renewal and proliferation to embryonic and adult stem cells, as they also express a number of key pluripotent transcription factors, including amongst others, NANOG, OCT3/4 and SOX2. Furthermore, since discreet groups of such stem cells are proposed to essentially drive tumourigenesis, they present as potential novel targets for cancer therapy. This study aimed to isolate a putative CSC population from the advanced colon adenocarcinoma cell lines HT29 and DLD1 and to assess the therapeutic effects of the epigenetic drugs Valproic acid and Zebularine on pluripotent gene expression.

Colon cancer, a disease in which malignant tumors form in the tissues of colon, is the first commonest cancer and the second leading cause of cancer-related deaths in Hong Kong. The standard treatment options for colon cancer include surgery and chemotherapy. However, multidrug resistance (MDR) develops in nearly all patients with colon cancer. In fact, most of the cancer-related deaths are due to chemotherapy failure caused by MDR, which occurs during the course of cancer progression and chemotherapy. Thus, the reversal of MDR plays an important role in the successful chemotherapy for colon cancer. This study investigated such a pharmacological action in reversing MDR in colon cancer cells by tanshinones, targeting the two common mechanisms responsible for MDR, i.e. overexpression of ATP-binding cassette (ABC) transporters and suppression of apoptosis. / Overexpression of P-glycoprotein (P-gp), one of the most important ABC transporters, can mediate the efflux of drugs out of cancer cells, leading to MDR and chemotherapy failure. The reversal of P-gp-mediated MDR by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone was evaluated in colon cancer cells. Bi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased the P-gp-mediated digoxin efflux in Caco-2 cells. The two tanshinones potentiated the cytotoxicities of doxorubicin and irinotecan in P-gp overexpressing colon cancer SW620 Ad300 cells. Moreover, these two tanshinones also increased intracellular accumulation of P-gp substrate in SW620 Ad300 cells, presumably by down-regulating P-gp mRNA and protein levels, as well as inhibiting P-gp ATPase activity. / Suppression of apoptosis can lead to MDR in cancer cells to anticancer agents with pro-apoptotic property. Hence, this study also investigated the circumvention of resistance to apoptosis in drug resistant colon cancer cells by cryptotanshinone and dihydrotanshinone, two potential MDR-reversing tanshinones. The drug resistant SW620 Ad300 cells were still sensitive to both cryptotanshinone and dihydrotanshinone in the promotion of cell death. When compared with the parental SW620 cells, the two tanshinones induced less apoptosis but more autophagy in the drug resistant cells. Further studies showed that cell viability was increased after inhibition of autophagy by siRNA interference or autophagy inhibitor. Thus, autophagy induced by the two tanshinones was pro-cell death in SW620 Ad300 cells, which could overcome resistance to apoptosis. / In addition, suppression of apoptosis can be caused by p53 defects/mutations, which were found in more than 50% of all human cancers. Our results also showed that apoptosis and autophagy induced by cryptotanshinone and dihydrotanshinone were independent of the status of p53 in colon cancer cells. The p53-independent cytotoxic actions of the two tanshinones could be useful in overcoming resistance to apoptosis in cancer cells caused by p53 defects/mutations. / Taken together, the current findings indicate a great potential of cryptotanshinone and dihydrotanshinone in the reversal of MDR caused by P-gp overexpression and suppression of apoptosis. They are promising candidates to be further developed as therapeutic agents in the adjuvant therapy for colon cancer, especially for the multidrug resistant cancer types. / 結腸癌是指形成在結腸組織的惡性腫瘤，在香港常見的癌症中排第一位，亦是香港排第二位的致死癌症。結腸癌的標準治療方案主要包括手術和化療。然而，多藥耐藥是結腸癌成功化療的一個障礙。事實上，大多數癌症引起的死亡都和在癌症的發展和化療的過程中產生的多藥耐藥有關。因此，多藥耐藥的逆轉對於結腸癌的成功化療非常重要。本研究旨在通過針對多藥耐藥兩種常見的機制ABC跨膜蛋白的過表達和抑制的細胞凋亡來探討丹參酮對結腸癌細胞多藥耐藥的逆轉。 / P-gp的過表達可介導藥物排出癌細胞，從而導致多藥耐藥和化療失敗。本研究評價了tanshinone I，tanshinone IIA，cryptotanshinone，dihydrotanshinone和miltirone對P-gp介導的結腸癌細胞多藥耐藥的逆轉。雙向轉運實驗表明，只有cryptotanshinone和dihydrotanshinone可以減少P-gp介導的digoxin外排。這兩個丹參酮可以增加doxorubicin和irinotecan在P-gp過表達的結腸癌SW620 Ad300細胞中的毒性。此外，這兩個丹參酮也增加P-gp底物在SW620 Ad300細胞內的積累，推測是通過下調P-gp的mRNA和蛋白水平，以及抑制P-gp的ATP酶活性。 / 抑制的細胞凋亡可導致腫瘤細胞對促凋亡的抗癌藥物产生多藥耐藥。因此，本研究也探討了cryptotanshinone和dihydrotanshinone能否克服結腸癌細胞的凋亡耐受。結果表明cryptotanshinone和dihydrotanshinone仍然能够杀死耐藥的SW620 Ad300細胞。當與SW620細胞相比，這兩個丹參酮在耐藥細胞中誘導的細胞凋亡較少，但自噬增多。進一步研究表明，這兩個丹參酮誘導的自噬是促進細胞死亡的，從而可以克服細胞的凋亡耐受。 / 此外，p53的缺陷/突變存在於50%以上的人類癌症中，并可以抑制細胞產生凋亡。結果表明，cryptotanshinone和dihydrotanshinone誘導的凋亡和自噬與p53在結腸癌細胞中的表達無關。這兩個丹參酮不依賴於p53的細胞毒性可以用於克服p53缺陷/突變引起的凋亡耐受。 / 綜上所述，本研究結果表明cryptotanshinone和dihydrotanshinone在逆轉P-gp的過表達和抑制的細胞凋亡引起的多藥耐藥中具有巨大潛力。它們可以進一步發展為有前途的治療劑并用於結腸癌的輔助治療，尤其是用於多藥耐藥的結腸癌。 / Hu, Tao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 163-182). / Abstracts also in Chinese. / Title from PDF title page (viewed on 06, December, 2016). / Hu, Tao. / 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.

Chan, Man Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-126). / Abstract also in Chinese. / Examination Committee List --- p.ii / Declaration --- p.iii / Acknowledgements --- p.iv / 摘要（Abstract in Chinese) --- p.vi / Abstract --- p.viii / List of Abbreviations --- p.x / List of Figures and Tables --- p.xii / Table of Content --- p.xiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Background of photodynamic therapy (PDT) --- p.2 / Chapter 1.1.1 --- History of PDT --- p.2 / Chapter 1.1.2 --- Photochemistry --- p.3 / Chapter 1.1.3 --- Principal stages of PDT --- p.5 / Chapter 1.1.4 --- Light sources of PDT --- p.6 / Chapter 1.2 --- Anti-tumor effect of PDT --- p.8 / Chapter 1.2.1 --- Mode of cell death --- p.8 / Chapter 1.2.2 --- PDT-induced anti-tumor immunity --- p.9 / Chapter 1.3 --- Clinical applications of PDT --- p.11 / Chapter 1.3.1 --- Photofrin® --- p.11 / Chapter 1.3.2 --- Clinical applications of PDT --- p.13 / Chapter 1.3.3 --- Challenges of PDT for clinical applications --- p.15 / Chapter 1.4 --- The development of new photosensitizers --- p.16 / Chapter 1.4.1 --- Targeted PDT --- p.16 / Chapter 1.4.2 --- Phthalocyanine --- p.18 / Chapter 1.5 --- Objective of my study --- p.21 / Chapter Chapter 2 --- Materials and Methods --- p.23 / Chapter 2.1 --- Synthesis of glucosylated silicon(IV) phthalocyanine (SiPcGlu) --- p.24 / Chapter 2.2 --- In vitro studies --- p.24 / Chapter 2.2.1 --- Cell line and culture conditions --- p.24 / Chapter 2.2.2 --- Photodynamic treatment --- p.25 / Chapter 2.2.3 --- Cell viability assay --- p.27 / Chapter 2.2.4 --- Light dose effect on the photocytotoxicity of SiPcGlu-PDT --- p.27 / Chapter 2.2.5 --- Determination of reactive oxygen species (ROS) production by SiPcGlu-PDT --- p.29 / Chapter 2.2.6 --- Effect of antioxidants on the photocytotoxicity of SiPcGlu-PDT --- p.29 / Chapter 2.2.7 --- Determination of ROS production after SiPcGlu-PDT --- p.30 / Chapter 2.2.8 --- Glucose competitive assay --- p.30 / Chapter 2.2.9 --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay --- p.30 / Chapter 2.2.10 --- DNA fragmentation analysis by gel electrophoresis --- p.31 / Chapter 2.2.11 --- Annexin-V & propidium iodide staining assay --- p.32 / Chapter 2.2.12 --- Subcellular localization studies --- p.33 / Chapter 2.2.13 --- Detection of mitochondrial superoxide production --- p.34 / Chapter 2.2.14 --- Assessment of mitochondrial membrane potential --- p.34 / Chapter 2.2.15 --- Caspase-3 activity assay --- p.35 / Chapter 2.2.16 --- "Western blot analyses for cytochrome c, caspase-3, PARP and glucose-regulated protein 78 (GRP78)" --- p.36 / Chapter 2.2.17 --- Ca2+ release from endoplasmic reticulum (ER) --- p.37 / Chapter 2.3 --- In vivo studies --- p.37 / Chapter 2.3.1 --- HT29 tumor-bearing nude mice model --- p.37 / Chapter 2.3.2 --- In vivo photodynamic treatment --- p.39 / Chapter 2.3.3 --- Biodistribution of SiPcGlu --- p.39 / Chapter 2.3.4 --- Assay for plasma enzyme activities --- p.40 / Chapter 2.4 --- Statistical analysis --- p.41 / Chapter Chapter 3 --- Results --- p.42 / Chapter 3.1 --- In vitro studies --- p.43 / Chapter 3.1.1 --- SiPcGlu-PDT induced cytotoxicity on HT29 cells --- p.43 / Chapter 3.1.2 --- Light dose effect on cytotoxicity by SiPcGlu-PDT --- p.46 / Chapter 3.1.3 --- SiPcGlu-PDT induced ROS production --- p.48 / Chapter 3.1.4 --- SiPcGlu-PDT induced cell death through Type I and II photoreactions --- p.48 / Chapter 3.1.5 --- ROS production after SiPcGlu-PDT --- p.51 / Chapter 3.1.6 --- Glucose competitive Assay --- p.55 / Chapter 3.1.7 --- SiPcGlu-PDT induced apoptosis in HT29 cells --- p.57 / Chapter 3.1.8 --- Subcellular localization of SiPcGlu --- p.61 / Chapter 3.1.9 --- SiPcGlu-PDT induced mitochondrial changes --- p.66 / Chapter 3.1.10 --- SiPcGlu-PDT induced caspase activation --- p.68 / Chapter 3.1.11 --- SiPcGlu-PDT increased expression of ER chaperone GRP78 --- p.72 / Chapter 3.1.12 --- SiPcGlu-PDT induced release of Ca2+ from ER --- p.72 / Chapter 3.2 --- In vivo studies --- p.75 / Chapter 3.2.1 --- In vivo photodynamic activities --- p.75 / Chapter 3.2.2 --- Tissue distribution of SiPcGlu --- p.77 / Chapter 3.2.3 --- Analysis of intrinsic toxicity --- p.77 / Chapter Chapter 4 --- Discussion --- p.80 / Chapter 4.1 --- Physical Properties of SiPcGlu --- p.81 / Chapter 4.2 --- In vitro studies --- p.82 / Chapter 4.2.1 --- SiPcGlu-PDT exhibits a high potency in killing HT29 cells --- p.82 / Chapter 4.2.2 --- ROS production is responsible for the cytotoxic effect of SiPcGlu-PDT --- p.83 / Chapter 4.2.3 --- SiPcGlu-PDT induced apoptosis in HT29 cells --- p.85 / Chapter 4.2.4 --- SiPcGlu is localized in various membranous organelles --- p.87 / Chapter 4.2.5 --- SiPcGlu-PDT induced mitochondria-mediated apoptosis --- p.89 / Chapter 4.2.6 --- SiPcGlu-PDT induced ER stress --- p.93 / Chapter 4.3 --- In vivo studies --- p.96 / Chapter 4.3.1 --- SiPcGlu failed to target to tumor tissues --- p.96 / Chapter 4.3.2 --- SiPcGlu-PDT induced retardation in tumor growth --- p.99 / Chapter 4.3.3 --- SiPcGlu is a safe photosensitizer for PDT --- p.101 / Chapter Chapter 5 --- Conclusion and Future Perspectives --- p.103 / Chapter 5.1 --- Conclusion --- p.104 / Chapter 5.2 --- Future Perspectives --- p.106 / Chapter 5.2.1 --- In vitro studies --- p.106 / Chapter 5.2.1.1 --- Lysosomal pathway to cell death --- p.106 / Chapter 5.2.2 --- In vivo studies --- p.107 / Chapter 5.2.2.1 --- Pharmacokinetic studies --- p.107 / Chapter 5.2.2.2 --- Eradication of HT29 tumor by repeated dose of SiPcGlu --- p.108 / Chapter 5.2.2.3 --- SiPcGlu-PDT-induced anti-tumor immunity --- p.108 / Chapter 5.2.2.4 --- Enhancement of tumor selectivity by conjugating with biomolecules --- p.109 / References --- p.110