There is some evidence to suggest that the pineal gland influences neoplastic growth. Either crude or partially purified pineal extracts have been used to treat malignant neoplasms in humans. More compelling evidence indicates that the pineal hormone melatonin, in addition to its well-known antireproductive effects, may also exert oncostatic effects particularly in animal models of human breast cancer. The purpose of this study was to examine a possible direct action of melatonin on the growth morphology and physiology of human breast cancer cells in vitro. Studies are described in which physiological concentrations of melatonin are shown to have markedly inhibitory effects directly on MCF-7 human breast cancer cells grown in culture. This antimitotic effect is not observed in MCF-7 cells at supra- or subphysiological concentrations of melatonin. This growth-inhibitory effect appears to be tissue specific since fibroblastic cells were not affected by melatonin. Other pineal indoles failed to inhibit the proliferation of this human breast cancer cell line, suggesting that this growth-inhibitory effect is specific for melatonin and is not a general characteristic shared among the family of pineal indoles. Reductions in media serum concentrations dramatically suppressed the response of cells to melatonin's inhibitory action. Serum values of 2.5 percent or lower resulted in a loss of melatonin's action as did growing the cells in serum-free medium or medium containing charcoal-treated serum. It appears that certain serum factors are necessary for these cells to respond to melatonin's antiproliferative action. Melatonin, when added to cells grown in media supplemented with 10 percent fetal calf serum decreased the synthesis of proteins and resulted in morphological alterations suggestive of a sublethal toxic injury. Melatonin appears to have a direct role in inhibiting the proliferation of breast cancer cells; however, the presence of melatonin per se does not seem to be the fundamental cause of this antimitotic action since no activity is observed when cells are propagated in media containing charcoal-treated fetal calf serum or serum-free medium. There appears to be a requirement for certain serum factors in this antiproliferative action. Two factors that have proved important in this process are the hormones estradiol and prolactin. (Abstract shortened with permission of author.)

Pancreatic Ductal Adenocarcinoma (PDAC) continues to have a dismally low survival rate due to late diagnosis and poor treatment options. Therefore, there is a need to understand the early stages and progression of the disease. PDAC is known to have two types of cells of origin: ductal cells or acinar cells. Since acinar-derived PDAC is thought to be the more malignant of the two, it was chosen as the focus of this work. Most studies of acinar cells as they relate to PDAC are accomplished by using animal models such as genetically engineered mouse models. While this method yields a large amount of insight into the progression of the disease and the role of specific genes, it has the drawbacks of being very time and resource intensive. The quicker and less costly alternative is <i>in vitro </i>culture. Specifically, here we have developed a microfluidic model which can incorporate a key aspect of the extracellular matrix (ECM), type I collagen, and mimics the 3D geometry of an <i>in vivo </i>acinus. Most attempts at <i>in vitro </i>culture have been limited by the fact that isolated acinar cells show a decrease in the amount of digestive enzymes they secrete as culture continues. For this reason, we are using a reprogrammed cancer cell line. These cells can be induced with doxycycline to express PTF1a, which allows the cells to adapt acinar characteristics, such as the production of digestive enzymes. We were able to successfully culture and induce PTF1a in these cells within our chip. We showed that the cells exhibit no invasion into the collagen matrix once PTF1a is expressed, thus eliminating a key aspect of cancer cell culture. The cells grown in the chip are confirmed to be producing PRSS2, the digestive enzyme trypsinogen. Collectively, this suggests that we have produced healthy acinar cells growing in the same configuration that they would <i>in vivo. </i>This has many applications in the study of pancreatic ductal adenocarcinoma, as we have developed way to culture reprogramed cancer cells as their benign precursors and maintain acinar characteristics <i>in vitro.</i> It will also have applications in the study of many other pancreatic diseases by providing an <i>in vitro</i> model of a healthy, functional acinus.

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. It is a multi-domain protein containing a Zinc finger, a RING finger, an Rb binding domain, a p53 binding domain and a novel N-terminal protein domain, the so called, Domain With No Name or DWNN. The RBBP6 gene encodes three isoforms of this particular protein. A common feature of all three isoforms of RBBP6 is the presence of the N-terminal DWNN domain. RBBP6 isoform 3 is comprised of the DWNN domain only. The DWNN itself has a ubiquitin-like fold, sharing 22% similarity with ubiquitin. It is likely that DWNN regulates intracellular levels of the two tumour suppressors, Rb and p53 through the ubiquitin-proteasome pathway and as such, DWNN may therefore play a role in the deregulation of cell cycle control in cancer cells. A mouse homologue, P2P-R of the gene has been implicated in mitotic apoptosis. The expression of DWNN, RBBP6 and their roles in the cell cycle, apoptosis and human cancer were investigated. RT-PCR and real-time PCR were used to determine the gene expression of DWNN and RBBP6 variants in human cancer cells. An anti-human DWNN antibody was characterized using both Western Blotting analysis and MALDI-TOF mass spectroscopy to determine whether the antibody specifically recognizes DWNN and RBBP6 isoforms, or if it recognizes other proteins. Western blotting was also used to determine the nature of the DWNN in human cell lines. A DWNN probe and the characterized anti-human antibody were used to localize DWNN and RBBP6 gene products at the mRNA and protein levels using ISH/FISH and Immunohistochemistry respectively. Cell labelling was also performed using this antibody to localize RBBP6 products in human cell lines. RNA interference and over-expression of DWNN and RBBP6 gene products was carried out to further investigate the role of RBBP6 products in the cell cycle, apoptosis and carcinogenesis. Cloned RT-PCR products of RBBP6 binding domains, the RING finger domain, pRb-binding and p53-binding domains in human cancers cell lines (Hek 293T, MCF7, HeLa and HepG2 cells) showed no mutations, but MCF-7 cells showed the lowest expression of the RBBP6. Real-time PCR and Western blotting analysis confirmed that MCF-7 cells express very little DWNN (RBBP6 isoform 3) and RBBP6 gene products when compared to Hek 293T, HeLa and HepG2 cells. It was also shown that the anti-human DWNN antibody recognizes the DWNN domain (RBBP6 isoform 3) and the larger RBBP6 isoforms. Using 2D gel electrophoresis and MALDI-TOF spectrometry, it was also found that DWNN is associated with other proteins namely, Recoverin and a hypothetical protein XP_002342450. This result suggested that DWNN may be a ubiquitin-like protein, which may be specific to these proteins in human cells. FISH and IHC demonstrated that the DWNN domain and its relatives are down-regulated in human cancers at both mRNA and protein levels, respectively. In contrast, however, cell staining showed that the expression of the DWNN gene products was high during the G2/Mitosis transition. Knocking-down the DWNN domain or over-expressing it did not sensitise the Hek 293T cells to Camptothecin (CPT)-induced apoptosis but rather slowed down cell growth. These results strongly suggest that the DWNN gene is likely to be involved in cell cycle control. Up-regulation in mitotic cells and down-regulation in cancers also implies that RBBP6 gene products may additionally be involved in cell cycle arrest. Moreover, down-regulation in human cancers particularly indicates that the loss of its function which correlates with loss of cell cycle control in this disease may be involved in the pathogenesis of cancer. This was confirmed by up-regulation of the DWNN in arsenic trioxide induced cell cycle arrested cells specifically at G2/M phase where a p53-dependent cell cycle arrest ensued. It is thus proposed that the DWNN may be implicated both as a p53 stabilizer and additionally as a G2/M progression regulator.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science Johannesburg, 2017 / Cancer is characterised by abnormal cell proliferation and is one of the leading causes of death in first world countries and the second leading cause in developing countries. In 2012 alone, over 14 million cases were reported and over 8 million deaths were attributed to cancer worldwide, with sub-Saharan Africa, especially South Africa having one of the highest oesophageal cancer rates in the world. An important aspect of cancer is the telomeres, which are 10-15kbp of TTAGGG DNA repeats in humans at the ends of chromosomes. These repeats are maintained by the enzyme telomerase. Up to 90% of all cancers show increased telomerase activity to overcome the "end-replication" problem in which the telomeres shorten after each cell division. This eventually leads to cellular senescence. Due to the high number of cancers relying on increased telomerase activity to bypass senescence, telomerase could be a viable target for anti-cancer therapies. The limiting factor of the multi-subunit telomerase enzyme is its telomerase reverse transcriptase component (hTERT). hTERT has also been shown to migrate to the mitochondria during times of high oxidative stress caused by reactive oxygen species (ROS). Here it confers protection to the mitochondria against ROS, potentially preventing the cell form undergoing apoptosis and reaching senescence. This can potentially be detrimental, as cells become damaged by the ROS and continue dividing. This could lead to further genetic damage. Metformin, a drug used for the treatment of type-2 diabetes, has been linked to lower incidences of cancer. The mode of action of metformin is not yet fully understood, however it is known that it affects the mitochondria. Since hTERT and metformin could co-localise, the drug may influence hTERT and potentially telomerase activity. This makes metformin an anticancer candidate to be used in conjunction with traditional anticancer therapies. To determine telomerase activity in metformin treated oesophageal carcinoma cells, qPCR based telomerase activity assays must be used. These assays can be very expensive and time consuming, so a faster and cheaper alternative would be beneficial. Therefore, the aim of this project was to alter and improve a nanoparticle based detection method for telomerase activity, by decreasing the time required to prepare the DNA functionalised nanoparticles as well as determining a more rapid method of data measurement, and compare it to conventional qPCR based techniques (TRAPeze RT Telomerase Activity Kit – Merck). Thereafter the effects of the metformin treatment on telomere dynamics, such as telomere length, telomerase activity and hTERT mRNA expression, in oesophageal squamous carcinoma cells were determined. Gold nanoparticles were synthesised and functionalised with thiolated-DNA (telomerase substrate). These functionalised particles were characterised using transmission electron microscopy. To assess telomerase activity the extracted protein was added to the functionalised nanoparticle solution and allowed to elongate the coupled DNA. A characteristic of gold nanoparticles is that the size of the particles as well as their proximity to one another determines the colour of the nanoparticle solution. Due to the steric hindrance caused by the now elongated DNA, a distinct colour change was observable. The change in absorption spectra of the nanoparticle solution was recorded after the enzyme elongated the substrate. This nanoparticle based assay was then compared to TRAPeze RT Telomerase detection kit (Merck-Millipore) as a positive control. Using the conventional qPCR based telomerase activity assay, it was found that metformin significantly decreased telomerase activity in oesophageal cancer cell lines, however this was not seen using the nanoparticle assay. A colour change was observed with the nanoparticle assay compared to the negative control reflecting detection of telomerase activity. However, no significant decrease in telomerase activity could be detected due to metformin treatment. More optimisation is required, however this technique has great potential, as nanoparticle based assays are also known for their high sensitivity. This technique is also far more rapid and significantly cheaper that the qPCR based method. The gold nanoparticle based telomerase activity assay could become an alternative to conventional qPCR based techniques. / MT2018

鈣粘蛋白是一類通過影響細胞粘附和細胞信號通路在腫瘤發生中起重要作用的細胞間粘附分子。鈣粘蛋白超家族包括經典鈣粘蛋白和非經典鈣粘蛋白，其中非經典鈣粘蛋白包含了原鈣粘蛋白。啟動子CpG甲基化調控下的基因沉默或表達下調是腫瘤發生中一個關鍵事件的觀點現已得到廣泛認可。一些鈣粘蛋白家族成員，如鈣粘蛋白-1/4/13（CDH1，CDH4，CDH13）被已有研究報導是受擬遺傳學調控沉默的功能性腫瘤抑制基因。本研究主要針對兩個鈣粘蛋白家族成員鈣粘蛋白-11（CDH11）和原鈣粘蛋白-10（PCDH10）進行腫瘤發生相關功能和機制的研究。 / CDH11位於雜合性缺失（LOH）經常發生而預示可能存在抑癌基因的染色體16q21-22區域，我們實驗室先前通過基因組芯片雜交技術（aCGH）對腫瘤細胞系的研究已發現它是該區域一個可能的抑癌基因。我們通過進一步的半定量反轉錄聚合酶鏈反應（RT-PCR）發現CDH11在正常組織和永生化正常上皮細胞中廣泛表達，但在各腫瘤細胞系中表達下降。甲基化特異性聚合酶鏈反應（MSP）和亞硫酸氫鹽處理的基因組測序（BGS）檢測到CDH11啟動子甲基化常發生于腫瘤細胞和腫瘤組織中。在CDH11表達缺失的腫瘤細胞中重新導入該基因的表達可顯著減少細胞克隆的形成，誘導細胞凋亡并抑制腫瘤細胞的遷移。通過更深入的機制研究，我們發現CDH11通過抑制Wnt/β-catenin信號通路發揮功能。 / 本研究的另一個鈣粘蛋白家族成員是PCDH10。之前我們實驗室的工作已經證實了PCDH10是一個在鼻咽癌和食管癌中受啟動子甲基化調控的抑癌基因,這裡我們主要研究它在大腸癌發病中的功能和機制。我們發現在PCDH10表達缺失的大腸癌細胞中重新導入PCDH10表達可顯著抑制腫瘤細胞的克隆形成，細胞遷移和幹細胞性。機制研究顯示PCDH10抑制Wnt/β-catenin和RhoA信號轉導通路，并進一步抑制腫瘤上皮細胞-間充質轉化（EMT）的過程，誘導幹細胞標記的下調。 / 綜上所述，本研究顯示CDH11和PCDH10兩個鈣粘蛋白家族成員在多種腫瘤中廣泛受甲基化調控失活，它們是重要的Wnt/β-catenin信號通路拮抗因素，可抑制腫瘤細胞的克隆形成和細胞遷移 / Cadherins are an important group of cell-cell adhesion molecules, which play crucial roles in tumorigenesis by affecting cell adhesion and cell signaling. Cadherin superfamily consists of classical cadherins and non-classical cadherins including protocadherins. It has been well recognized that silencing or downregulation of tumor suppressor genes (TSGs) by promoter CpG methylation is a critical event in human tumorigenesis. Some cadherin family members, such as CDH1, CDH4, CDH13, have been reported as functional TSGs silenced through epigenetic regulation. In this study, I mainly focus on the function and mechanism studies of two cadherin members-Cadherin 11(CDH11) and Protocadherin 10 (PCDH10). / CDH11 is located in 16q21-22, a region with frequent loss of heterozygosity (LOH), indicating the presence of candidate TSG. Previously, our lab also identified CDH11 as a candidate TSG through array-CGH of tumor cell lines. I further found by semi-quantitative RT-PCR that CDH11 was broadly expressed in normal tissues while frequently downregulated in multiple tumor cell lines, but not in immortalized normal epithelial cells. Methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) detected frequent promoter methylation of CDH11 in tumor cell lines and primary tumor samples. Ectopic expression of CDH11 dramatically reduced tumor cell clonogenecity, induced tumor cell apoptosis and inhibited tumor cell migration. By further mechanism study, I found that CDH11 is a negative inhibitor of Wnt/β-catenin signaling pathway. / Another cadherin family protein which I chose to study is PCDH10. Previously our lab identified PCDH10 as a TSG by promoter methylation in nasopharyngeal and esophageal carcinomas. I studied the function and mechanism of PCDH10 in the pathogenesis of colon cancer. I found ectopic expression of PCDH10 strongly suppressed colon tumor cell clonogenecity, migration and stemness. Moreover, I found that PCDH10 repressed Wnt/β-catenin and RhoA signaling, thus further inhibited the epithelial-to-mesenchymal transition (EMT) of tumor cells and downregulated stem cell markers. / In summary, this study demonstrates two cadherin family members CDH11 and PCDH10, as important antagonists to Wnt/β-catenin signaling pathway, suppress tumor cell clonogenecity, migration, and are also frequently inactivated by epigenetic mechanism in multiple tumors. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Yanjiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 84-95). / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of Contents --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / List of Publications --- p.xvi / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.1.1 --- General introduction about cancer --- p.1 / Chapter 1.1.2 --- Oncogenes and TSGs --- p.3 / Chapter 1.1.3 --- Cancer mechanism models --- p.3 / Chapter 1.2 --- Cancer Epigenetics --- p.5 / Chapter 1.2.1 --- DNA methylation --- p.6 / Chapter 1.2.2 --- DNA methylation and gene silencing --- p.7 / Chapter 1.2.3 --- DNA methylation and cancer --- p.7 / Chapter 1.2.4 --- Clinical implications of DNA methylation --- p.8 / Chapter 1.3 --- Cadherins --- p.10 / Chapter 1.3.1 --- General introduction of cadherin superfamily --- p.10 / Chapter 1.3.2 --- Cadherin classification --- p.10 / Chapter 1.3.3 --- Cadherin and cancers --- p.12 / Chapter 1.3.4 --- Cadherin switch and EMT in cancer --- p.16 / Chapter 1.4 --- Wnt/β-catenin signaling pathway and cancer --- p.16 / Chapter 1.4.1 --- Wnt/β-catenin signaling pathway --- p.16 / Chapter 1.4.2 --- Wnt/β-catenin signaling pathway in cancer --- p.18 / Chapter 1.4.3 --- Epigenetic silencing of Wnt/β-catenin signaling --- p.20 / Chapter 1.4.4 --- Wnt/β-catenin signaling pathway in CRC --- p.21 / Chapter Chapter 2 --- Aims of the Study --- p.22 / Chapter Chapter 3 --- Materials and Methods --- p.25 / Chapter 3.1 --- Cell lines and tissue samples --- p.25 / Chapter 3.1.1 --- Cell lines --- p.25 / Chapter 3.1.2 --- Maintenance of cell lines --- p.25 / Chapter 3.1.3 --- Drug treatment of cell lines --- p.26 / Chapter 3.1.4 --- Normal and primary tissues --- p.26 / Chapter 3.1.5 --- Total RNA extraction --- p.27 / Chapter 3.1.6 --- Genomic DNA extraction --- p.28 / Chapter 3.2 --- Gene expression analysis --- p.29 / Chapter 3.2.1 --- Reverse transcription (RT) --- p.29 / Chapter 3.2.2 --- Semi-quantitative RT-PCR --- p.30 / Chapter 3.3 --- Methylation Analysis --- p.32 / Chapter 3.3.1 --- CpG island prediction --- p.32 / Chapter 3.3.2 --- Sodium bisulfite treatment of genomic DNA --- p.33 / Chapter 3.3.3 --- Methylation-specific PCR (MSP) --- p.33 / Chapter 3.3.4 --- Bisulfite Genomic Sequencing (BGS) --- p.34 / Chapter 3.4 --- Construction of expression plasmids --- p.36 / Chapter 3.4.1 --- Construction of CDH11 expression vector --- p.36 / Chapter 3.4.2 --- Construction of PCDH10 expression plasmid --- p.38 / Chapter 3.4.3 --- Plasmid extraction --- p.39 / Chapter 3.5 --- Plasmid transfection --- p.41 / Chapter 3.6 --- Subcellular localization --- p.42 / Chapter 3.7 --- Function analyses --- p.43 / Chapter 3.7.1 --- Colony formation assay --- p.43 / Chapter 3.7.2 --- Wound healing assay --- p.44 / Chapter 3.8 --- Mechanism exploration --- p.44 / Chapter 3.8.1 --- Protein extraction and western-blot --- p.44 / Chapter 3.8.2 --- Dual-luciferase reporter assay --- p.47 / Chapter 3.9 --- Statistical analysis --- p.48 / Chapter Chapter 4 --- CDH11 functions as a tumor suppressor via modulating Wnt/β-catenin signaling and is frequently downregulated by promoter methylation --- p.49 / Chapter 4.1 --- The CpG island of CDH11 gene promoter --- p.50 / Chapter 4.2 --- CDH11 expression profile in normal tissues --- p.50 / Chapter 4.3 --- Frequent silencing of CDH11 by promoter methylation in multiple tumors --- p.51 / Chapter 4.4 --- Restoration of CDH11 expression by pharmacologic demethylation --- p.53 / Chapter 4.5 --- Frequent CDH11 methylation in primary tumors --- p.54 / Chapter 4.6 --- Function studies --- p.56 / Chapter 4.6.1 --- Ectopic expression of CDH11 inhibited tumor cell clonogenecity --- p.57 / Chapter 4.6.2 --- CDH11 induced tumor cell apoptosis --- p.57 / Chapter 4.6.3 --- CDH11 inhibited tumor cell migration --- p.58 / Chapter 4.7 --- CDH11 antagonized Wnt/β-catenin signaling --- p.59 / Chapter 4.8 --- Discussion --- p.60 / Chapter Chapter 5 --- Epigenetic inactivated tumor suppressor PCDH10 exerts tumor suppressive functions through modulating Wnt/β-catenin signaling and cell stemness in colon cancer --- p.66 / Chapter 5.1 --- PCDH10 was broadly expressed in normal tissues and frequently silenced in CRC cell lines --- p.67 / Chapter 5.2 --- Promoter methylation mediated PCDH10 silencing in CRC cell lines --- p.68 / Chapter 5.3 --- Frequent PCDH10 methylation in CRC primary tumors --- p.69 / Chapter 5.4 --- PCDH10 was located in cytoplasm and membrane --- p.70 / Chapter 5.5 --- Function Studies --- p.71 / Chapter 5.5.1 --- PCDH10 inhibited clonogenicity of tumor cells --- p.71 / Chapter 5.5.2 --- PCDH10 suppressed tumor cell mobility --- p.72 / Chapter 5.6 --- Mechanism exploration of PCDH10 in CRC --- p.72 / Chapter 5.6.1 --- PCDH10 antagonized Wnt/β-catenin signaling --- p.72 / Chapter 5.6.2 --- PCDH10 negatively regulated EMT and stemness of tumor cells --- p.74 / Chapter 5.6.3 --- PCDH10 inhibited RhoA signaling --- p.75 / Chapter 5.7 --- Discussion --- p.75 / Chapter Chapter 6 --- Conclusions and Future studies --- p.80 / Chapter 6.1 --- Conclusions --- p.80 / Chapter 6.2 --- Future studies --- p.82 / Reference List --- p.84

Lee Mei-sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 123-133). / Abstracts in English and Chinese. / ACKOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / ABSTRACT in Chinese (摘要） --- p.iv / LIST OF FIGURES AND TABLES --- p.vi / LIST OF ABBREVIATIONS --- p.viii / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1 --- Discovery of polyphyllin D in pharmaceutical study --- p.1 / Chapter 1.2 --- Structure of Polyphyllin D --- p.4 / Chapter 1.3 --- Origin of Polyphyllin D --- p.4 / Chapter 1.4 --- Polyphyllin D in antitumor activities analysis --- p.6 / Chapter 1.5 --- Characteristic of Polyphyllin D --- p.9 / Chapter 1.6 --- General properties of Saponin --- p.9 / Chapter 1.6.1 --- Membrane-disrupting property --- p.10 / Chapter 1.6.2 --- Hemolytic Property --- p.10 / Chapter 1.7 --- Antitumor properties of saponins --- p.11 / Chapter 1.7.1 --- Induction of apoptosis by saponins --- p.11 / Chapter 1.7.2 --- Induction of cell cycle arrest by saponins --- p.12 / Chapter 1.7.3 --- Saponins with structure similar to polyphyllin D are found with antitumor activity --- p.13 / Chapter 1.8 --- Human breast cancer --- p.14 / Chapter 1.8.1 --- Incidence of Breast Cancer --- p.14 / Chapter 1.8.2 --- Classification of Breast Cancer --- p.16 / Chapter 1.8.3 --- Role of estrogen and estrogen receptor in breast cancer --- p.16 / Chapter 1.8.4 --- Today treatment in Breast Cancer --- p.18 / Chapter 1.8.4.1 --- Hormonal Therapy --- p.18 / Chapter 1.8.4.1.1 --- Tamoxifen --- p.19 / Chapter 1.8.4.2 --- Chemotherapy --- p.20 / Chapter 1.8.4.2.1 --- Doxorubicin (Adriamycin) --- p.20 / Chapter 1.9 --- The Hypothesis and the aim my project --- p.22 / Chapter CHAPTER 2: --- Materials and methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Cell Lines --- p.24 / Chapter 2.1.1.1 --- MCF-7 --- p.24 / Chapter 2.1.1.2 --- MDA-MB-231 --- p.25 / Chapter 2.1.1.3 --- HS68 --- p.25 / Chapter 2.1.1.4 --- WRL-68 --- p.25 / Chapter 2.1.2 --- Culture Medium --- p.26 / Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.26 / Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-free Medium) --- p.27 / Chapter 2.1.2.3 --- Dulbecco's modified Eagle's medium --- p.27 / Chapter 2.2 --- Reagent and Buffers --- p.28 / Chapter 2.2.1 --- Regents for DNA fragmentation Assay --- p.28 / Chapter 2.2.2 --- Regents for Western Blot Analysis --- p.29 / Chapter 2.2.3 --- Reagent for Two Dimensional Electrophoresis Analysis --- p.31 / Chapter 2.2.3.1 --- Protein Preparation --- p.31 / Chapter 2.2.3.2 --- First dimension Electrophoresis --- p.31 / Chapter 2.2.3.3 --- Second dimension Electrophoresis --- p.32 / Chapter 2.2.3.4 --- Silver staining --- p.32 / Chapter 2.2.4 --- Reagent for ln-gel digestion --- p.33 / Chapter 2.2.4.1 --- Destaining --- p.33 / Chapter 2.2.4.2 --- Digestion --- p.33 / Chapter 2.2.4.3 --- Desalting of the peptide mixture for MS analysis --- p.33 / Chapter 2.2.5 --- Reagent for flow cytomertry analysis --- p.34 / Chapter 2.2.6 --- Reagent for immunofluorescent staining --- p.34 / Chapter 2.2.7 --- Reagent for Primary mouse speenocytes and macrophages preparation --- p.34 / Chapter 2.2.8 --- Reagent for Cell Cytotoxicity Assay --- p.35 / Chapter 2.2.9 --- Reagent for in vivo study --- p.35 / Chapter 2.2.10 --- Reagent for Estrogen Receptor Competitive Assay --- p.35 / Chapter 2.3 --- Chemicals --- p.36 / Chapter 2.4 --- Methods --- p.38 / Chapter 2.4.1 --- Polyphyllin D preparation --- p.38 / Chapter 2.4.1.1 --- in vitro application --- p.38 / Chapter 2.4.1.2 --- in vivo application --- p.39 / Chapter 2.4.2 --- Treatment of polyphyllin in vitro --- p.39 / Chapter 2.4.3 --- MTT assay --- p.39 / Chapter 2.4.4 --- Trypan Blue Exclusion Assay --- p.40 / Chapter 2.4.5 --- Analysis of Cell-Cycle Phase Distribution by Flow cytometry with PI staining --- p.41 / Chapter 2.4.6 --- Estrogen Receptor competitive Assay --- p.41 / Chapter 2.4.7 --- Nucleosome Detection Assay --- p.42 / Chapter 2.4.8 --- Quantification of Apoptosis by Flow Cytometry with Annexin V ´ؤ PI Staining --- p.42 / Chapter 2.4.9 --- Assessment of the Change in Mitochondrial Membrane Potential (Δφ m) --- p.43 / Chapter 2.4.10 --- Western Blotting Analysis --- p.44 / Chapter 2.4.10.1 --- Protein Extraction --- p.45 / Chapter 2.4.10.2 --- Protein concentration Determination --- p.47 / Chapter 2.4.10.3 --- SDS acrylamide gel electrohphoresis --- p.47 / Chapter 2.4.10.4 --- Electroblotting of Protein --- p.47 / Chapter 2.4.10.5 --- Probing of Proteins with Antibodies --- p.48 / Chapter 2.4.10.6 --- Enhanced Chemiluminescence (ECL) Assay --- p.48 / Chapter 2.4.11 --- Two Dimensional Electrophoretic Analysis --- p.49 / Chapter 2.4.11.1 --- Protein preparation --- p.50 / Chapter 2.4.11.2 --- First dimensional electrophoresis --- p.51 / Chapter 2.4.11.2.1 --- Rehydration of IPG strips --- p.51 / Chapter 2.4.11.2.2 --- IEF with IPGphor --- p.51 / Chapter 2.4.11.2.3 --- Running IPG strips --- p.52 / Chapter 2.4.11.2.4 --- Eauilibration of the IPG strip --- p.52 / Chapter 2.4.11.3 --- Second dimensional electrophoresis Equilibration of the IPG strip --- p.52 / Chapter 2.4.11.4 --- Visualization of the 2D gel by Silver staining --- p.53 / Chapter 2.4.11.5 --- Computer analysis of the 2d gel image --- p.54 / Chapter 2.4.12 --- Protein identification with Matrix assisted laser desorption- ionization Time-of-flight mass spectrometry (MALDI-TOF) --- p.54 / Chapter 2.4.12.1 --- In gel tryptic digestion --- p.54 / Chapter 2.4.12.2 --- Desalting of the peptide mixtures --- p.55 / Chapter 2.4.12.3 --- Database Searching --- p.55 / Chapter 2.5 --- Statisitc Analysis --- p.55 / Chapter CHAPTER 3: --- Cytotoxic effects of polyphyllin D / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.2 --- Cytotoxic activities on human breast cancer cell lines --- p.58 / Chapter 3.2.1 --- In MCF-7 cells --- p.53 / Chapter 3.2.2 --- In MDA-MB-231 cells --- p.58 / Chapter 3.3 --- Cytotoxic activities on human normal cell lines --- p.63 / Chapter 3.3.1 --- "Human normal liver cell line, WRL-68" --- p.63 / Chapter 3.3.2 --- "Human skin fibroblast cell line, HS-68" --- p.63 / Chapter 3.4 --- Cytotoxic activities on primary culture --- p.65 / Chapter 3.4.1 --- Primary mouse spleenocytes --- p.65 / Chapter 3.5 --- Conclusion --- p.59 / Chapter CHAPTER 4: --- Induction of apoptosis / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.2 --- Induction of apoptosis on MCF-7 cells --- p.71 / Chapter 4.2.1 --- Nucleosome formation by ELISA assay --- p.71 / Chapter 4.2.2 --- Phosphatidylserine Extemalization by flow cytometrial study --- p.73 / Chapter 4.3 --- Induction of apoptosis on MDA-MB-231 cells --- p.75 / Chapter 4.3.1 --- Apoptotic peak by flow cytometrial study --- p.75 / Chapter 4.3.2 --- Phosphatidylserine Extemalization by flow cytometrial study --- p.78 / Chapter 4.4 --- Conclusion --- p.80 / Chapter CHAPTER 5 --- : Induction of apoptosis via mitochondrial pathway / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.2 --- Mitochondrial Membrane Depolarization by flow cytometrial study --- p.82 / Chapter 5.2.1 --- In MCF-7 cells --- p.84 / Chapter 5.2.2 --- In MDA-MB-231 cells --- p.84 / Chapter 5.3 --- Alternation of mitochondrial protein expression by western blot analysis on MCF-7 and MDA-MB-231 cells --- p.87 / Chapter 5.4 --- Activation of caspase9 --- p.90 / Chapter 5.5 --- Conclusion --- p.92 / Chapter CHAPTER 6 --- : Antitumor activities independent of estrogen receptor / Chapter 6.1 --- Introduction --- p.93 / Chapter 6.2 --- Completion assay on Estrogen Receptor binding --- p.95 / Chapter 6.3 --- Expression level of Estrogen receptor --- p.97 / Chapter 6.4 --- Conclusion --- p.99 / Chapter CHAPTER 7 --- : Proteomic detection of intracellular changes / Chapter 7.1 --- Introduction --- p.100 / Chapter 7.2 --- Differential Protein expression profile of MCF-7 cells --- p.101 / Chapter 7.3 --- Elevated HLA-A antigen expression --- p.104 / Chapter 7.3.1 --- Mass fingerprinting with MALDI-TOF-MS --- p.104 / Chapter 7.3.2 --- Flow cytometrial analysis with immunofluoresent staining --- p.107 / Chapter 7.4 --- Conclusion --- p.109 / Chapter CHAPTER 8 --- : Discussion / Chapter 8.1 --- Polyphyllin D is Invaluable for further Investigation --- p.110 / Chapter 8.2 --- Induction of common mitochondrial pathway --- p.111 / Chapter 8.3 --- Induction of Fas receptor apoptotic pathway is unknown --- p.112 / Chapter 8.4 --- Polyphyllin D is Caspase-3 independent --- p.113 / Chapter 8.5 --- MDA-MB-231 is more sensitive to polyphyllin D --- p.113 / Chapter 8.6 --- Antitumor effects regardless of induction of apoptosis --- p.114 / Chapter 8.7 --- Advances in antitumor effect by MHC-1 antigen up-regulation --- p.114 / Chapter 8.8 --- Implication of polyphyllin D in up-regulating MHC-1 antigen --- p.115 / Chapter 8.9 --- Future aspect --- p.117 / Chapter 8.10 --- Conclusion --- p.121 / Chapter CHAPTER 9 : --- References --- p.122

根據文獻研究記載，化療藥物可誘導癌細胞的凋亡，這是公認的化療療法的主要治療效果。作為一種程式性細胞死亡，積累的實驗證據表明，誘導所致的細胞凋亡是可逆轉的。這就引出了對於細胞凋亡恢復及其調節機制的相關問題。 / 在這項研究中，我們證明了在質膜不對稱的散失和半胱天冬酶（caspase）啟動後，HeLa細胞的凋亡的啟動可逆轉。我們發現，除了被廣泛研究的抗增殖作用外，金雀異黃素(genistein)可抑制細胞凋亡的復蘇。即時定量PCR發現抗凋亡基因MDM2和XIAP在凋亡逆轉過程中表達水準上調，金雀異黃素可抑制其表達水準的上調。金雀異黃素，MDM2蛋白抑制劑和XIAP抑制劑的利用，造成復原細胞內持續的半胱天冬酶活性和增強的細胞死亡效果。然而，半胱天冬酶抑制劑並不能挽救金雀異黃素的抑制作用。流式細胞儀的研究表明，金雀異黃素可以導致凋亡逆轉細胞持久磷脂醯絲氨酸（PS）外化和逆轉細胞的細胞壞死。抑制半胱天冬酶活性將金雀異黃素的主要作用轉移到壞死效果。這些結果揭示了金雀異黃素抑制細胞凋亡逆轉的兩個可能的機制。 / 金雀異黃素能維持現有的細胞凋亡信號從而增強細胞凋亡。它也可以破壞凋亡恢復過程，導致繼發性壞死。金雀異黃素對於細胞凋亡逆轉的抑制可與常規化療相結合，以提高治療結果. / It is well documented that chemotherapeutical agents could induce apoptosis of cancer cells, which is recognized as a major treatment effect of chemotherapy. Accumulating evidence indicates that chemopreventive agents like soybean isoflavone genistein could potentiate the antitumor effect of chemotherapeutic drugs both in vivo and in vitro. The mechanistic basis of this augmentation effect by genistein remains to be fully elucidated. / In this study, we demonstrated while low-concentration ethanol stressed cancer cells could recover, the presence of genistein promoted the cell death of stressed cancer cells that displayed apoptotic features. In HeLa cells, quantitative real-time PCR revealed the up-regulation of anti-apoptotic genes MDM2 and XIAP during the recovery process, and genistein suppressed their expression. The application of genistein, MDM2 inhibitor and XIAP inhibitor to the recovering HeLa cells caused persistent caspase activity and enhanced cell death. However, the death-promoting effect of genistein was not rescued by caspase inhibitor. Flow cytometry study indicated that genistein treatment could lead to persistent phosphatidylserine (PS) externalization and necrotic events in the recovering HeLa cells. Caspase activity inhibition shifted the major effect of genistein to secondary necrosis. / These results suggested two possible mechanisms through which genistein promoted cell death in stressed HeLa cells. Genistein could maintain the existing apoptotic signal to enhance apoptotic cell death. It could also disrupt the recovering process in caspase-independent manner, which lead to secondary necrosis. These effects may account for the enhanced antitumor effect of chemotherapeutic drugs when they were combined with genistein. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Xin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 79-90). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in also in Chinese. / Cover Page / Statement --- p.i / Thesis Committee members --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / Table of contents --- p.vi / List of abbreviations --- p.ix / List of figures and tables --- p.xi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction to general cancer biology --- p.1 / Chapter 1.1.1 --- Overview of cancer --- p.1 / Chapter 1.1.1.1 --- Classification of cancer --- p.1 / Chapter 1.1.1.2 --- Risk factors of carcinogenesis --- p.2 / Chapter 1.1.1.3 --- Cancer prevention and therapies --- p.4 / Chapter 1.1.2 --- Models of cancer development --- p.6 / Chapter 1.1.2.1 --- Multistage model of carcinogenesis --- p.6 / Chapter 1.1.2.2 --- Colorectal cancer as an example of multistep / multigene carcinogenesis --- p.7 / Chapter 1.1.2.3 --- Driving force for cancer development --- p.9 / Chapter 1.1.3 --- Properties of cancer cells --- p.11 / Chapter 1.2 --- Apoptosis and its roles in cancer development and treatment --- p.14 / Chapter 1.2.1 --- Overview of apoptosis --- p.14 / Chapter 1.2.2 --- Molecular mechanism of apoptosis --- p.15 / Chapter 1.2.3 --- Positive and negative regulation of apoptosis --- p.18 / Chapter 1.2.4 --- Apoptotic defects in cancer development --- p.20 / Chapter 1.2.5 --- Apoptosis in cancer treatment --- p.23 / Chapter 1.3 --- The reversibility of apoptotic events --- p.25 / Chapter 1.4 --- Genistein and its relevance to cancer therapy --- p.27 / Chapter 1.5 --- Objectives of the study --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Cancer cell lines --- p.30 / Chapter 2.1.2 --- Cell culture media and additives --- p.30 / Chapter 2.1.3 --- Biochemical kits --- p.30 / Chapter 2.1.4 --- Chemicals and reagents --- p.30 / Chapter 2.1.5 --- Antibodies --- p.31 / Chapter 2.1.6 --- Primers used for quantitative real-time PCR --- p.32 / Chapter 2.1.7 --- Buffers and solutions --- p.32 / Chapter 2.2 --- Methods and procedures --- p.33 / Chapter 2.2.1 --- Cell culture establishment and cryopreservation --- p.33 / Chapter 2.2.2 --- Living cell staining and imaging --- p.34 / Chapter 2.2.3 --- MTT cell viability assay --- p.34 / Chapter 2.2.4 --- BrdU cell proliferation assay --- p.35 / Chapter 2.2.5 --- LDH cytotoxicity assay --- p.35 / Chapter 2.2.6 --- Quantitative real-time PCR --- p.36 / Chapter 2.2.7 --- Western blotting --- p.37 / Chapter 2.2.8 --- Annexin V/ Propidium Iodide Assay --- p.38 / Chapter 2.2.9 --- Trypan Blue Dye Exclusion Assay --- p.39 / Chapter 2.2.10 --- Cleaved-Caspase 3 Immunostaining --- p.39 / Chapter 2.2.11 --- Statistical Analysis --- p.39 / Chapter Chapter 3 --- Results --- p.40 / Chapter 3.1 --- Low concentration ethanol stressed cancer cells displayed apoptotic features and the stressed cell could recover after stress removal --- p.40 / Chapter 3.1.1 --- Morphological changes and apoptotic marker activation in low concentration ethanol stress --- p.40 / Chapter 3.1.2 --- In situ study of morphological changes and caspase 3 activation in HeLa --- p.44 / Chapter 3.2 --- Genistein promoted the cell death of stressed cancer cells at non-cytotoxic concentration towards unstressed cells --- p.46 / Chapter 3.2.1 --- Dose-dependent response of genistein on stressed and unstressed cells --- p.46 / Chapter 3.2.2 --- In HeLa cells, genistein suppressed the recovery from stress treatment at non-cytotoxic concentration --- p.48 / Chapter 3.2.3 --- Genistein promoted both apoptosis and necrosis in stressed cells. . --- p.49 / Chapter 3.3 --- Genes involved in the recovery from stress treatment were influenced by genistein --- p.53 / Chapter 3.3.1 --- Stressed HeLa cells were more sensitive to the inhibition of de novo synthesis --- p.53 / Chapter 3.3.2 --- Expression profiles of genes involved in recovery and the influence of genistein --- p.55 / Chapter 3.4 --- Like genistein, MDM2 and XIAP inhibitor potentiated the cell death and caused persistent caspase-3 activity in stressed cells --- p.58 / Chapter 3.4.1. --- Stressed HeLa cells were much more sensitive to the inhibition of XIAP and MDM2 --- p.58 / Chapter 3.4.2 --- The presence of inhibitor at non-cytotoxic concentration to unstressed cells suppressed the recovery of the stressed cells --- p.60 / Chapter 3.4.3 --- Genistein, MDM2 inhibitor and XIAP inhibitor caused persistent apoptotic signals in recovering cells. --- p.61 / Chapter 3.5 --- The death-promoting effect by genistein could be caspase-independent --- p.64 / Chapter 3.6 --- Caspase activity abrogation shifted genistein’s action profile --- p.66 / Chapter Chapter 4 --- Discussion and prospect --- p.70 / Chapter 4.1 --- The apoptotic features were induced by low concentration ethanol stress --- p.70 / Chapter 4.2 --- The apoptotic features caused by ethanol stress were reversible --- p.71 / Chapter 4.3 --- Genistein showed death-promoting effects on the recovering cells --- p.72 / Chapter 4.4 --- The genes (XIAP and MDM2) that were involved in the recovery process may function to terminate apoptotic signal --- p.73 / Chapter 4.5 --- Genistein suppressed the upregulation of anti-apoptotic genes and promoted the expression of pro-apoptotic genes --- p.74 / Chapter 4.6 --- The XIAP and MDM2 activity were essential for the recovery from stress --- p.75 / Chapter 4.7 --- Caspase inhibition increased the secondary necrosis in recovering cells with genistein treatment --- p.76 / Chapter 4.8 --- Hypothetic mechanism of genistein’s inhibitory effect on the recovery of stressed cells --- p.77 / Chapter 4.9 --- Summary and prospects --- p.78 / Reference list --- p.79

This research utilizes Electrical Impedance Spectroscopy, a technique classically used for electrochemical analysis and material characterization, as the basis for a non-destructive, label-free assay platform for three dimensional (3D) cellular spheroids. In this work, a linear array of microelectrodes is optimized to rapidly respond to changes located within a 3D multicellular model. In addition, this technique is coupled with an on chip micro-pH sensor for monitoring the environment around the cells. Finally, the responses of both impedance and pH are correlated with physical changes within the cellular model. The impedance analysis system realized through this work provides a foundation for the development of high-throughput drug screening systems that utilize multiple parallel sensing modalities including pH and impedance sensing in order to quickly assess the efficacy of specific drug candidates. The slow development of new drugs is mainly attributed to poor predictability of current chemosensitivity and resistivity assays, as well as genetic differences between the animal models used for tests and humans. In addition, monolayer cultures used in early experimentation are fundamentally different from the complex structure of organs in vivo. This requires the study of smaller 3D models (spheroids) that more efficiently replicate the conditions within the body. The main objective of this research was to develop a microfluidic system on a chip that is capable of deducing viability and morphology of 3D tumor spheroids by monitoring both the impedance of the cellular model and the pH of their local environment. This would provide a fast and reliable method for screening pharmaceutical compounds in a high-throughput system.

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human homologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs'. Edd null mice and hyd mutants display embryonic lethality. EDD is also a multifunctional HECT family E3 ubiquitin protein-ligase, with reported roles in both progesterone action and the DNA damage response. To investigate the possible involvement of EDD in human cancer, several cancer types were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus. AI of the EDD locus was most frequent in the serous subtype of ovarian cancer (16/22, 73%) and common in other cancers, including breast cancer (31%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers. These data imply a potential role for EDD in cancer progression. However, depletion of EDD from cells in culture by RNA interference had very little effect on proliferation and cell survival. To identify EDD-regulated pathways, transcript analysis was performed on EDD-depleted cells. The results suggested that EDD modulates cell-cell communication and the actin cytoskeleton. Consistent with transcript analysis, depletion of EDD from two normal breast cell lines (HMEC-184 and MCF-10A) resulted in altered cell morphology, with decreased cell-cell contacts. This was concurrent with altered beta-catenin (an integral component of adherens junctions) at cell-cell contacts, which was also observed in the developing blood vessels of Edd null mice. Interestingly, total cellular beta-catenin levels were not affected. Furthermore, EDD depletion resulted in a decrease in expression of the cytoskeletal regulators twinfilin and R-RAS, with a simultaneous decrease in MAPK (ERK1 and ERK2) activity. Consistent with disruption of adherens junctions, EDD-depleted mammary acini lost tissue coordination and polarity. These data provide a significant advance in our knowledge of EDD, both in its role in regulating the organisation of cells into higher structures and its potential role in the development of cancer. This has relevance to an understanding of embryonic development and the role of tissue homeostasis in cancer progression.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xiv, 139 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 129-138).