M.A. / The aim of this study was to ascertain the coping methods of women in long term follow-up of breast cancer treatment. Furthermore, personality traits that deal with the spectrum of positive affectivity were introduced to determine whether these impact on women's appraisal of their situation and their subsequent choice of coping mechanism. Thus, a process approach to exploring coping strategies and a goal-attainment conceptualization of hope were used to determine whether hope is associated with coping appraisal in the long term follow-up of breast cancer treatment. Furthermore, high hope women were expected to use more problem focused coping methods and low hope women were expected to use more emotion focused coping skills. Women in cancer remission who attend yearly or six-monthly check-ups at the Johannesburg hospital were approached to complete the questionnaire and brief interview. Although the study did not confirm that low hope and high hope women use different kinds of coping strategies, the predicted relationship between hope and challenge appraisals was supported by significant correlations. However, it was found that hope may be analogous to positive affect, thus indicating the need for further validation of the Hope Scale. Finally, it was concluded that breast cancer need not be seen as a devitalising disease and that there are a variety of coping strategies which can be utilized to enhance patient's positive emotional state. The women in this study use the emotion focused coping skill of positive reappraisal which concentrates on the possibilities for mastery and growth that inhere in their long term follow-up treatment. Moreover, women are extremely positive and hopeful in their daily outlook and while this personality trait seems to suggest that denial is at play, it is more likely that women in long term remission have a strong belief in their own personal qualities and future. Women in this study choose to distance themselves from the implicit trauma of the threat of recurrence in favour of an active belief in their personal resilience to overcome any stressful event or outcome.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in Diagnostic Radiology Johannesburg, 2013 / Palpable solid breast masses that are circumscribed and not calcified on mammogram or ultrasound are probably benign. There is controversy therefore, whether these deserve tissue diagnosis. More data is required to determine whether short term follow up can replace the need for biopsy. Benign appearing lesions greater than 3cm in diameter on ultrasound continue to undergo biopsy due to fear that a malignancy or phyllodes tumour might be missed. Published research reflects patients from Europe and North America, and no relevant data from Africa exists. AIM: This study aims to determine the histological spectrum of sonographically benign lesions greater than 3cm, which were biopsied, in our local population (majority of black patients) and to determine whether biopsy is indicated based on the local cancer risk. The study also aims to characterise the results by age and population group as well as correlate the histological result with the size of the lesion on ultrasound, the HIV status, family history and the seniority of the examining radiologists. MATERIALS AND METHODS: A retrospective descriptive study of biopsy results of sonographically benign breast masses was undertaken using biopsy procedural recording sheets. . The size of the lesions (continuous variables) mean with standard deviations was determined. The prevalence of lesions was expressed as a percentage. Other categorical variables were summarized as frequency and percentage. The vi histological spectrum of the lesions was determined. The HIV status and family history of the patients as well as the seniority of the reviewing radiologist was assessed. A Krusskal Wallis test and separate logistic regression analysis was used. RESULTS: A total of 68 patients (below 40 years of age) were included from a total of 13112 patients (of all ages) seen between 2007 and the end of 2010. 73 lesions were identified (65 benign and 8 malignant). The prevalence of benign lesions was 89.7%. .The prevalence of malignant lesions was 10.29%.There was little evidence to support lesion size for predicting histology (p value = 0.22) or benignity. There was little evidence that the family history and HIV status were significant. CONCLUSION: There was a high prevalence (10.29%) of malignancies in lesions classified by ultrasound as benign. The size of the lesion did not correlate with histological subtype or whether the lesion was benign or malignant. Training of sonographers, standardization of technique for established users and double reading, may produce a different result, as both junior and senior radiologists mistook malignant lesions for benign ones on ultrasound. Repeating this research using double reading after training may demonstrate whether there is a true higher prevalence of malignancy in ultrasonically benign breast lesions in our community. Until then, routine biopsy of these lesions is recommended.

A simple method to verify the total treatment time generated by the treatment planning system (TPS) when the CONTURA MLB or the SAVI applicator are used for APBI treatments has been developed. The method compares the time generated by the TPS to a predicted time, calculated by inserting parameters obtained from the TPS in equations generated in this Thesis. The equations were generated by linearly fitting data from clinical cases that had been treated using the Contura MLB or the SAVI applicator at the Lynn Cancer Institute of the Boca Raton Regional Hospital. The parameters used were the PTV coverage, Air Kerma Strength, Balloon Volume (Contura data fit) and Evaluation PTV (SAVI data fit). As an outcome of this research, it is recommended that the plan should be reevaluated when the percent difference between the generated and the predicted times exceeds 5% for the Contura MLB, or 10% for the SAVI. / by Andreas Kyriacou. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Breast cancer is the most common cancer among females and it is the leading cause of death in mid-age women. Epidemiological studies indicate that Asian women have a lower incidence of breast cancer compared with their counterparts in the West, which soy consumption has been suggested as a contributory factor. Soy and soy-based food contain a rich amount of phytoestrogens, which are suggested to be protective against cancer. Cyclooxygenase (COX) is the rate-limiting enzyme for the conversion of arachidonic acid to prostaglandins. Recent studies have revealed that up-regulation of cyclooxygenase-2 (COX-2), an isoform of COX, plays an important role in tumorigenesis and metastasis. COX-2 may facilitate carcinogenesis in a number of means, may include altering cell proliferation and apoptosis, enhancing angiogenesis and suppressing immune surveillance. Clinical examinations of breast cancer specimens indicated that COX-2 is overexpressed. In the present study, we investigated the effect of flavonoids on COX-2 expression in human breast cells. / Our results showed that daidzein and its metabolite eqoul, genistein, butein, isoliquiritigenin (ILN) and apigenin could inhibit phorbol 12-myristate 13-acetate (PMA)-induced COX-2 expression in breast cells MCF-7 and MCF-10A. The inhibitory effects were in concentration-dependent manners. Real-time PCR and western blot analysis showed that the flavonoids suppressed the induced mRNA and protein expression. Suppression could be observed in concentration as low as 0.1 muM. Luciferase reporter assay indicated that the inhibition was at the gene transactivation level. Further investigation using truncated hCOX-2 promoter plasmids revealed that the AP-1 site (-67/-61) and cyclic AMP response element (CRE) site (-59/-53) on hCOX-2 promoter were responsible for the suppression. Electrophoretic mobility shift assay results further confirmed that the flavonoids acted through inhibiting AP-1/CREB DNA binding to suppress the expression. / To examine the possible upstream signal transduction pathways involved, inhibitors of protein kinase A (PKA), protein kinase (PKC) and mitogen activated protein kinase (MAPK) were employed. Reporter gene assay revealed a possible involvement of ERK1/2 MAP kinase in AP-1 and/or CRE activation of hCOX-2 promoter. Taken together, these results suggested that the phytochemicals down-regulated PMA-induced COX-2 expression by counteracting AP-1 and CRE sites via the modulation of MAPK pathway. The findings might have significant implications in the chemopreventive and chemotherapeutic applications of flavonoids in breast cancer. / Lau, Tak Yi. / "December 2007." / Adviser: Lai Kwok Leung. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4734. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 142-164). / 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.

by Ng Ping-chung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 161-179). / List of Abbreviation / Abstract / Acknowledgements / Table of contents / Chapter 1. --- Introduction / Chapter 1.1 --- Hormone and carcinogenesis --- p.1 / Chapter 1.2 --- Estrogen and carcinogenesis --- p.3 / Chapter 1.2.1 --- Carcinogenesis and endogenous sex hormone status --- p.3 / Chapter 1.2.2 --- Etiology of breast cancer --- p.3 / Chapter 1.2.2.1 --- Epidemiology --- p.3 / Chapter 1.2.2.2 --- Hormonal factors --- p.5 / Chapter 1.2.2.3 --- Genetic predisposition --- p.8 / Chapter 1.2.2.4 --- Influence of diet --- p.8 / Chapter 1.2.3 --- Hormonal therapy --- p.18 / Chapter 1.2.3.1 --- Anti-estrogen --- p.18 / Chapter 1.2.3.2 --- Progestins --- p.21 / Chapter 1.2.3.3 --- Aromatase inhibitor --- p.22 / Chapter 1.2.3.4 --- GnRH analogue therapy --- p.26 / Chapter 1.3 --- Estrogen pool --- p.26 / Chapter 1.4 --- Estrogen receptor --- p.30 / Chapter 1.4.1 --- General features of estrogen receptor and action mechanism --- p.30 / Chapter 1.4.2 --- Anti-estrogen binding site (AEBS) --- p.31 / Chapter 1.4.3 --- Physiological consideration --- p.32 / Chapter 1.4.3.1 --- Uterus: uterotrophic responses --- p.32 / Chapter 1.4.3.2 --- "Progesterone, the physiological estrogen antagonist" --- p.34 / Chapter 1.5 --- The role of growth factors and steroid hormones in breast cancer cell --- p.35 / Chapter 1.6 --- Alternate cytotoxic action of TAM --- p.37 / Chapter 1.7 --- In vitro models utilised in breast cancer study --- p.38 / Chapter 1.8 --- Current development of anti-estrogen --- p.39 / Chapter 1.9 --- Background about yuehchukene (YCK) --- p.41 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Studies using whole animals --- p.47 / Chapter 2.1.1 --- Uterotrophic assay in rats --- p.47 / Chapter 2.1.2 --- Anti-implantation assay in rats --- p.48 / Chapter 2.1.3 --- Vaginal smear in mice --- p.49 / Chapter 2.2 --- Studies using breast cancer cells --- p.49 / Chapter 2.2.1 --- MCF-7 cell culture --- p.49 / Chapter 2.2.1.1 --- Measurement of cell number --- p.50 / Chapter 2.2.1.1.1 --- Cell count with haemocytometer --- p.50 / Chapter 2.2.1.1.2 --- Cell number estimated by DNA content in culture using Hoechst33258 --- p.51 / Chapter 2.2.1.1.3 --- Cell number estimated by [3H]-thymidine incorporation --- p.52 / Chapter 2.2.1.1.4 --- Preparation of dextran coated charcoal stripped serum --- p.52 / Chapter 2.2.2 --- MDA-MB-231 cell culture --- p.53 / Chapter 2.3 --- Studies using steroid receptors --- p.54 / Chapter 2.3.1 --- Rat uterine estrogen receptor --- p.54 / Chapter 2.3.2 --- Mice uterus and vaginal estrogen receptor --- p.55 / Chapter 2.3.3 --- MCF-7 cell estrogen receptor --- p.55 / Chapter 2.3.3.1 --- MCF-7 whole cell estrogen receptor binding --- p.55 / Chapter 2.3.3.2 --- Cytosolic estrogen receptor preparation from MCF-7 cell --- p.57 / Chapter 2.3.4 --- Progesterone receptor binding in MCF-7 cell --- p.57 / Chapter 2.3.5 --- Rat hepatic anti-estrogen binding site (AEBS) --- p.58 / Chapter 2.3.6 --- Estrogen receptor content estimation by enzyme immunoassay --- p.58 / Chapter 2.4 --- Enzyme studies related to estrogen metabolism --- p.60 / Chapter 2.4.1 --- Rat uterine ornithine decarboxylase (ODC) --- p.60 / Chapter 2.4.2 --- Rat hepatic ethoxyresorufin O-deethylase (EROD) --- p.60 / Chapter 2.4.3 --- Rat hepatic estradiol-2-hydroxylase --- p.62 / Chapter 2.4.4 --- MCF-7 cell estradiol-2-hydroxylase --- p.62 / Chapter 2.4.5 --- Human placental microsomal aromatase activity --- p.63 / Chapter 2.5 --- Enzymatic studies related to signal transduction --- p.64 / Chapter 2.5.1 --- Inhibition of Protein Kinase C activity of MCF-7 cell and protein phosphorylation --- p.64 / Chapter 2.5.2 --- Inhibition of calmodulin activation of cyclic nuleotide phosphodiesterase --- p.66 / Chapter 2.6 --- "Preparation of Pre-YCK, crude-YCK and post-YCK fractions" --- p.67 / Chapter 2.7 --- Preparation of Indole-3-carbinol acid condensation product (I3Ca) --- p.71 / Chapter 2.8 --- Studies on TCP series of YCK analogues --- p.71 / Chapter 2.9 --- List of test compounds --- p.75 / Chapter 2. 10 --- List of radio-ligands --- p.77 / Chapter 2.11 --- Miscellaneous reagents related to cell culture --- p.78 / Chapter 2.11.1 --- Culture medium --- p.78 / Chapter 2.11.2 --- Fetal calf serum --- p.78 / Chapter 2.11.3 --- Penicillin-streptomycin powder --- p.78 / Chapter 2.11.4 --- Phosphate buffer saline --- p.78 / Chapter 2.12 --- "Solvents, chemical and scintillants" --- p.78 / Chapter 3. --- Result / Chapter 3.1 --- Rat uterotrophic response with EE2 and YCK --- p.80 / Chapter 3.2 --- Mice vaginal cornification with estradiol (E2) and YCK --- p.83 / Chapter 3.3 --- Human breast cancer cell culture --- p.86 / Chapter 3.3.1 --- MCF-7 cell growth with YCK --- p.86 / Chapter 3.3.2 --- MCF-7 cell growth with YCK analogues and other related compounds --- p.91 / Chapter 3.3.3 --- MDA-MB-231 cell culture --- p.100 / Chapter 3.4 --- Receptor Binding --- p.100 / Chapter 3.4.1 --- Rat uterine estrogen receptor --- p.100 / Chapter 3.4.2 --- Mice uterine and vaginal estrogen receptor --- p.103 / Chapter 3.4.3 --- MCF-7 whole cell and cytosolic estrogen receptor --- p.103 / Chapter 3.4.4 --- MCF-7 cell progesterone receptor --- p.107 / Chapter 3.4.5 --- Rat hepatic anti-estrogen binding sites (AEBS) --- p.111 / Chapter 3.5 --- Enzyme activities related to estrogen metabolism --- p.111 / Chapter 3.5.1 --- Rat uterine ornithine decarboxylase (ODC) --- p.111 / Chapter 3.5.2 --- Rat hepatic estradiol-2-hydroxylase and ethoxyresorufin O-deethylase --- p.114 / Chapter 3.5.3 --- MCF-7 cell estradiol-2-hydroxylase --- p.121 / Chapter 3.5.4 --- Human placenta and MCF-7 cell aromatase --- p.126 / Chapter 3.6 --- Enzyme activities related to signal transduction --- p.126 / Chapter 3.6.1 --- Protein kinase C inhibition in vitro --- p.126 / Chapter 3.6.2 --- Calmodulin-dependent phosphodiesterase inhibitory actions in vitro --- p.131 / Chapter 3.7 --- Studies on TCP series of YCK analogues --- p.131 / Chapter 4. --- Discussion / Chapter 4.1 --- Estrogenicity of YCK --- p.140 / Chapter 4.2 --- Estrogenicity of YCK correlates with estrogen receptor (ER) binding --- p.141 / Chapter 4.3 --- Attenuation by YCK --- p.142 / Chapter 4.3.1 --- Attenuation by YCK on estrogen induced uterotrophic activity --- p.142 / Chapter 4.3.2 --- Attenuation by YCK on mice vaginal cornification with estradiol and YCK --- p.142 / Chapter 4.3.3 --- Attenuation by YCK on MCF-7 cell growth --- p.143 / Chapter 4.3.4 --- Attenuation of YCK on ornithine decarboxylase (ODC) induced by estrogen --- p.144 / Chapter 4.4 --- Deviation between YCK potency and RBA --- p.145 / Chapter 4.5 --- Estrogen inhibition action of YCK via non receptor binding mechanism --- p.148 / Chapter 4.6 --- Protein kinase C/ calmodulin-dependent phosphodiesterase inhibitor --- p.152 / Chapter 4.7 --- Progesterone receptor --- p.154 / Chapter 4.8 --- Aromatase inhibitor? --- p.155 / Chapter 4.9 --- Posssible mechanism for the attenuation of estrogenic action by YCK --- p.157 / Chapter 4.10 --- TCP series of YCK analogues --- p.158 / Chapter 4.11 --- Future works --- p.159 / Chapter 5. --- Reference --- p.161 / Appendix / Appendix 1 YCK analogues / Appendix 2 Structure of compounds mentioned in this thesis

Introduction: Basal-like (BL) breast cancer is an aggressive sub-type of breast cancer for which there is no targeted systemic therapy. C-Met is a receptor tyrosine kinase implicated in breast cancer. Clinical trials assessing the efficacy of anti-c-Met therapy are underway, yet few studies have analysed the clinical significance of c-Met expression and/or activation in breast cancer, in particular whether there is a correlation with molecular sub-type. The aims of this study are: 1) to establish the clinical significance of c-Met expression in invasive breast cancer, 2) evaluate the novel proximity ligation assay (PLA) as a method of measuring c-Met activation and 3) address the effect of hepatocyte growth factor (HGF)-mediated c-Met phosphorylation on migration and protein expression in cell lines representative of the BL sub-type. Methods: Immunohistochemistry for c-Met was performed on 1455 cases of breast cancer using tissue microarray (TMA) technology. The PLA was performed on TMAs constructed from 181 breast cancers. C-Met expression and the PLA product were correlated with clinico-pathological parameters and survival. The effects of HGF on cell migration and protein expression were assessed using migration assays, western blots and immunofluorescent studies. Results: C-Met expression was independently associated with BL breast cancer (odds ratio = 6.44, 95% confidence interval (CI) = 1.74-23.78, p = 0.005) and reduced overall survival (hazard ratio = 1.81, 95% CI = 1.07-3.06), p = 0.026). The PLA signal was not associated with molecular sub-type or survival. HGF stimulation was associated with a significant increase in BL cell migration (p < 0.01) but no evidence of epithelial-mesenchymal transition was observed. Conclusion: My findings suggest BL breast cancer patients should be included in future trials of anti-c-Met therapy. Further work is necessary to establish the prognostic utility of the PLA as a measure of c-Met activation and the mechanisms driving HGF-mediated cell migration.

Breast cancer is the most common cancer in the UK and Basal-like breast cancer (a highly aggressive subtype) accounts for approximately 8-22% of all cases depending on ethnicity. Unlike most human malignancies and indeed other PAM50 breast cancer subtypes, the vast majority of Basal-like tumours are positive for wild type p16. This p16 signature is associated with a particularly poor prognosis and p16-positive Basal-like breast cancer remains the most clinically challenging subtype and is the focus of this project. Pro-senescence therapies are gaining momentum as attractive strategies for the treatment of those breast cancers with current unmet clinical need. To identify targets for pro-senescence therapy in p16-positive Basal-like breast cancer, a genome‐wide siRNA screen and two subsequent validation screens using two p16-positive cancer cell lines were performed. Screening revealed 20 siRNAs that induced senescence within both cancer cell lines. Strikingly, 11 of these 20 siRNAs targeted ribosomal proteins, implicating disrupted ribosomal biosynthesis in senescence activation in p16-positive Basal-like breast cancer. Importantly, subsequent experiments in normal human mammary epithelial cells established that specific ribosomal protein knockdown is well tolerated by normal cells. Analysis of the METABRIC data set showed a high degree of ribosomal dysregulation in Basal-like tumours and revealed that all 11 ribosomal hits identified were frequently overexpressed in p16-positive Basal-like breast cancers. Kaplan Meier analysis confirmed that elevated expression of six of the 11 ribosomal proteins correlates with a reduced overall survival in these women, further supporting a role for these proteins as drivers of disease. These six ribosomal hits, associated with the poorest patient survival, were prioritised for further validation. Senescence induction was found to be highly stable, and associated with dramatic changes to nucleolar morphology, reminiscent of the nucleolar signature observed upon premature senescence induction in normal human mammary epithelial cells. In addition, siRNA rescue experiments indicated that senescence initiation is dependent on p16 and p21 expression and is accompanied by p16 nuclear translocation and p21 degradation. Further, ribosomal protein silencing in MDA-MB-231 cells (p16-null Basal-like breast cancer cell line) resulted in a 'death-like' phenotype, partially dependent on p21 expression suggesting that, within a cancer context, ribosomal protein silencing may induce a differential response depending on the status of p16. In conclusion, it is proposed that these six ribosomal candidates may form the basis of a novel pro-senescence therapy for p16-positive Basal-like breast cancer. They may also represent novel prognostic biomarkers for this disease subset and may help to improve disease stratification and future directed personalised therapies.

Metastasis is the most fearsome aspect of breast cancer (BC) a common disease in women, because it drives mortality. Although BC can invade almost any organ, it is most often found to invade the bone (31-79%), the brain (3-12%), the liver (8-18%) and the lung (11-13%). The site of distant metastasis is often associated with cause of death and length of survival. This dissertation examines whether the presence of select lifestyle and clinical factors can predict metastatic spread to the lung and/or the liver for a particular woman with advanced breast cancer. A systematic review of the literature identified tobacco use as a risk factor for lung metastasis in women with BC and suggested that obesity, hormone replacement therapy prior to BC diagnosis, hormonal therapy post diagnosis, and post-mastectomy radiation therapy may have an impact on this association. The review also uncovered that liver disease (i.e. hepatic steatosis, chronic hepatitis B infection, cirrhosis) is associated with the occurrence of liver metastasis in patients with colorectal cancer and that hyperglycemic and oxidative stress conditions as well as alcohol consumption were found to be associated with liver metastasis in colorectal or BC patients. We conducted a retrospective hospital-based case-control study of the association of select lifestyle and clinical factors with metastases detected in the lung and the liver among women diagnosed with stages II-IV BC and seen at the Columbia University Medical Center from 2008 to 2013. Select relevant clinical variables were extracted from the hospital patient charts and lifestyle factors from patients’ responses to a questionnaire developed for the purposes of this research. We examined whether smoking and / or post-mastectomy radiation therapy to the breast and/or the chest area were associated with an increased risk of 1st site lung metastasis in our sample of women with metastatic BC. We found that lifestyle factors such as smoking history or BMI at diagnosis did not affect the likelihood of 1st site lung metastasis in our sample of women. We also investigated whether a history of alcohol intake or chronic liver disease was associated with risk of developing a 1st site liver metastasis. Our analyses suggested that lifestyle factors such as alcohol intake or obesity might not affect the likelihood of 1st site liver metastasis in women with metastatic BC. We also report that a history of chronic liver disease significantly increased the odds of 1st site liver metastasis. Given our findings around adjuvant post mastectomy radiation therapy and chronic liver disease, we suggest collecting adjuvant treatment or relevant comorbid information in larger cohort studies. A better understanding of the relationship between these factors and the sites of metastasis has the potential to increase our understanding of the metastatic process. If we can find ways to identify women at high risk of metastatic disease, or develop preventive or therapeutic measures against lung or liver metastasis, we can hope to reduce mortality from metastases.

C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and prostate cancer stem cells. In breast cancer, CXCR4 signalling via stromal cell-derived factor-1 (SDF-1) has been reported to be a mediator of metastasis, and is linked to poor prognosis. However its role in normal and malignant breast stem cell function has not been investigated. Anoikis-resistant (AR) cells were collected from mammosphere culture from 2 immortalised (MCF10A, 226L) and 3 malignant (MCF7, T47D, SKBR3) breast cell lines. For all cell lines, AR cells had a significantly higher mammosphere forming efficiency (MFE) than unsorted cells. The AR cells of the normal cell lines also demonstrated increased formation of 3D structures using the Matrigel assay. In vivo, MCF7 and T47D AR cells demonstrated increased capacity to form tumours compared with unsorted cells. This suggests that AR cells are enriched for normal and malignant breast stem cells. We performed an Agilent custom gene microarray and demonstrated up-regulation of CXCR4 mRNA expression in AR cells. CXCR4 protein expression was also higher in AR cells, shown by flow cytometry. The effects of AMD3100 (CXCR4 antagonist) and SDF-1 (CXCR4 ligand) on stem cell activity were investigated in the mammosphere assay. In the normal cell lines, SDF-1 significantly reduced MFE and this decrease was rescued by AMD3100. Incubation with AMD3100 increased MFE in the estrogen receptor positive breast cancer cell lines (MCF7 and T47D) and patient-derived metastatic tumour samples. AMD3100 reduced the self-renewal of T47D cells, as assessed by second generation mammospheres. MCF7 cells were retro-virally transfected to over-express CXCR4 or sorted for CXCR4 cell surface expression. Mammosphere formation was significantly increased in CXCR4+ and CXCR4 over-expressing cells compared with CXCR4- and parental cells. There was a greater reduction in self-renewal following AMD3100 treatment in the CXCR4 over-expressing cells compared with parental cells. AMD3100 has been shown to have an agonistic effect on the novel chemokine receptor CXCR7, a scavenging receptor for SDF-1. All cell lines demonstrated cell surface expression of CXCR7, measured by flow cytometry and mRNA expression. Potential interactions between CXCR4, CXCR7 and SDF-1 must be considered in future investigation of the role of CXCR4 signalling. Our data establish that CXCR4 signalling has contrasting effects on normal and malignant breast stem cell activity. CXCR4 influences self-renewal of malignant stem cells which may account for its role in tumorigenesis. CXCR4 signalling may be important in tumour formation at the sites of metastases as well as in cell migration. Its role in stem cell migration merits further investigation. In conclusion, CXCR4-targeted therapy, alongside current standards of care, may improve breast cancer outcomes.

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