Tesis sobre el tema "Estrogen Therapeutic use"

[Truncated abstract] Our understanding of the actions of oestrogen on the vasculature has recently been questioned following the results of large clinical trials revealing a negative effect of hormone replacement therapy (HRT) on cardiovascular disease (CVD) risk amongst postmenopausal women. It is important to determine how a hormone with numerous positive effects on intermediate pathways of atherosclerosis fails to offer cardioprotection. Further investigation into the actions of oestrogen in the vasculature may add to our current understanding of the pathogenesis of atherosclerosis and oestrogen biology. The primary aim of this thesis was to investigate involvement of the oestrogen receptors (ERs) in atherosclerotic CVD and to provide further insight into the actions of oestrogen on the vasculature by studying the actions of oestrogen on the regulation of an oestrogen-responsive gene within human vascular cells. Following confirmation of ERa and ERß expression at the RNA and protein level in human aorta sections, correlations of receptor expression with age and atherosclerosis were examined. Significantly strong negative relationships of ERa, androgen receptor (AR), and progesterone receptor (PR) with age in both males and females were detected. No trend was detected between ERß expression and age. These findings suggest that the receptor-mediated actions of hormones in the vasculature may change with age. Further, this thesis compared for the first time sex hormone receptor expression in normal and adjacent atherosclerotic aortic tissue providing a critical assessment of receptor differences due to atherosclerosis. Results revealed reductions of all hormone receptors in early atherosclerotic versus normal aorta tissue. ... These results suggest that the 3'-UTR SNPS may have more of an influence on carotid thickening when oestrogen levels are lower, suggesting the importance of both genetic variation of the ERß gene and oestrogen status on carotid thickening. Finally, this was the first study to investigate oestrogen-induced regulation of angiotensinogen (AGT), a candidate gene for CVD, in human vascular cells. Oestrogen influenced AGT transcription in a cell specific manner. The overall influence of oestrogen on AGT transcription in the vasculature is unknown. This thesis adds to the knowledge of oestrogen and atherosclerosis by suggesting the involvement of the sex hormone receptors (ERa, ERß, PR and AR) in atherosclerosis, presenting ERß as a potentially important candidate gene for atherosclerosis, revealing interactions between estrogen status and associations of ERß SNPs with carotid thickening, and demonstrating vascular cell-specific actions of oestrogen on the regulation of a candidate gene for CVD. These factors may have contributed to the lack of cardio-protection following HRT, as revealed by large clinical trials.

The effectiveness of estrogen replacement therapy in the prevention of postmenopausal osteoporosis has led to its current widespread use throughout the United States and much of Western Europe, and recently, clinical correlations between circulating androgen levels and structural bone integrity have been presented. Nevertheless, the biochemical mechanism through which estrogens and androgens act to protect and maintain bone has remained unclear. One possibility is that these hormones directly modulate the activity of cells responsible for bone formation. Therefore, studies were conducted to examine the effects of sex steroids on human osteoblast-like cells. In the first set of experiments, a finite human cell line was established from trabecular bone explants obtained from a 48 year-old woman. These cells, designated BG688, were characterized as osteoblast-like in phenotype using several independent criteria. In addition to classical osteoblast markers, BG688 cells also possess approximately 2400 high affinity (K(d) = 0.45nM) 17-β estradiol (E₂) binding sites per cell. The binding of E₂ to a subset of these sites was specific. BG688 cells were also shown to respond to a physiological concentration (10nM) of E₂, which elicits pleiotropic changes in several mRNA levels including a 2-fold increase in the steady state concentration of α₁(I)-procollagen mRNA. These results indicate that human osteoblast-like cells respond to E₂ via a receptor mediated mechanism, but that, unlike the reproductive tissues, osteoblasts are a less sensitive target. In the second series of experiments, the effects of androgenic hormones on the osteoblast-like, human osteosarcoma cell line, HOS TE85 were evaluated. Employing radiolabelled dihydrotestosterone (DHT), 2800 saturable, high-affinity (K(d) = 0.66nM) androgen binding sites were detected per HOS TE85 cell. Androgen binding was specific. The expression of androgen receptors in HOS TE85 cells was further substantiated by Northern analysis. Physiological concentrations of DHT and testosterone decreased HOS TE85 cell proliferation. This finding suggests that androgens may also play a role in osteoblast differentiation. In support of this hypothesis, treatment with testosterone enhanced the abundance of both α₁ (I)-procollagen mRNA and transforming growth factor- β mRNA. The non-aromatizable androgen DHT also elicited an increase in the steady state concentration of α₁(I)-procollagen mRNA. The findings presented herein are significant within the field of bone cell biology in that they demonstrate that osteoblasts are a target cell for the action of sex steroids, via their cognate, high-affinity receptors. These results also have important implications within the broader context of bone pathophysiology in that they suggest a direct modulation of bone forming and bone remodeling activity by sex steroids.

Resumo: A decisão da terapêutica hormonal no tratamento do cancro da mama baseiase na determinação do receptor de estrogénio alfa por imunohistoquímica (IHC). Contudo, a presença deste receptor não prediz a resposta em todas as situações, em parte devido a limitações do método IHC. Investigámos se a expressão dos genes ESR1 e ESR2, bem como a metilação dos respectivos promotores, pode estar relacionada com a evolução desfavorável de uma proporção de doentes tratados com tamoxifeno assim como com a perda dos receptores de estrogénio alfa (ERα) e beta (ERß). Amostras de 211 doentes com cancro da mama diagnosticado entre 1988 e 2004, fixadas em formalina e preservadas em parafina, foram utilizadas para a determinação por IHC da presença dos receptores ERα e ERß. O mRNA total do gene ESR1 e os níveis específicos do transcrito derivado do promotor C (ESR1_C), bem como dos transcritos ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 foram avaliados por Real-time PCR. Os promotores A e C do gene ESR1 e os promotores 0K e 0N do gene ESR2 foram investigados por análise de metilação dos dinucleotidos CpG usando bisulfite-PCR para análise com enzimas de restrição, ou para methylation specific PCR. Atendendo aos resultados promissores relacionados com a metilação do promotor do gene ESR1, complementamos o estudo com um método quantitativo por matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) suportado pelo software Epityper para a medição da metilação nos promotores A e C. Fez-se a avaliação da estabilidade do mRNA nas linhas celulares de cancro da mama MCF-7 e MDA-MB-231 tratadas com actinomicina D. Baixos níveis do transcrito ESR1_C associaram-se a uma melhor sobrevivência global (p = 0.017). Níveis elevados do transcrito ESR1_C associaram-se a uma resposta inferior ao tamoxifeno (HR = 2.48; CI 95% 1.24-4.99), um efeito mais pronunciado em doentes com tumores de fenótipo ERα/PgR duplamente positivo (HR = 3.41; CI 95% 1.45-8.04). A isoforma ESR1_C mostrou ter uma semi-vida prolongada, bem como uma estrutura secundária da região 5’UTR muito mais relaxada em comparação com a isoforma ESR1_A. A análise por Western-blot mostrou que ao nível da 21 proteína, a selectividade de promotores é indistinguivel. Não se detectou qualquer correlação entre os níveis das isoformas do gene ESR2 ou entre a metilação dos promotores do gene ESR2, e a detecção da proteína ERß. A metilação do promotor C do gene ESR1, e não do promotor A, foi responsável pela perda do receptor ERα. Estes resultados sugerem que os níveis do transcrito ESR1_C sejam usados como um novo potencial marcador para o prognóstico e predição de resposta ao tratamento com tamoxifeno em doentes com cancro da mama. Abstract: The decision of endocrine breast cancer treatment relies on ERα IHC-based assessment. However, ER positivity does not predict response in all cases in part due to IHC methodological limitations. We investigated whether ESR1 and ESR2 gene expression and respective promoter methylation may be related to non-favorable outcome of a proportion of tamoxifen treated patients as well as to ERα and ERß loss. Formalin-fixed paraffin-embedded breast cancer samples from 211 patients diagnosed between 1988 and 2004 were submitted to IHC-based ERα and ERß protein determination. ESR1 whole mRNA and promoter C specific transcript levels, as well as ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 transcripts were assessed by real-time PCR. ESR1 promoters A and C, and ESR2 promoters 0N and 0K were investigated by CpG methylation analysis using bisulfite-PCR for restriction analysis, or methylation specific PCR. Due to the promising results related to ESR1 promoter methylation, we have used a quantification method by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) together with Epityper software to measure methylation at promoters A and C. mRNA stability was assessed in actinomycin D treated MCF-7 and MDA-MB-231 cells. ERα protein was quantified using transiently transfected breast cancer cells. Low ESR1_C transcript levels were associated with better overall survival (p = 0.017). High levels of ESR1_C transcript were associated with non-favorable response in tamoxifen treated patients (HR = 2.48; CI 95% 1.24-4.99), an effect that was more pronounced in patients with ERα/PgR double-positive tumors (HR = 3.41; CI 95% 1.45-8.04). The ESR1_C isoform had a prolonged mRNA half-life and a more relaxed 5’UTR structure compared to ESR1_A isoform. Western-blot analysis showed that at protein level, the promoter selectivity is undistinguishable. There was no correlation between levels of ESR2 isoforms or ESR2 promoter methylation and ERß protein staining. ESR1 promoter C CpG methylation and not promoter A was responsible for ERα loss. We propose ESR1_C levels as a putative novel marker for breast cancer prognosis and prediction of tamoxifen response.

This study analyzed the effects of estrogen replacement therapy (ERT), ethnicity, marital status, education level, maternal status and financial security on the perceived life satisfaction of women over fifty. Information was collected from 125 subjects at an independent school district. The instrument was adapted from a life satisfaction scale originally developed by B. Neugarten. Eight demographic items included ERT use, age, menopause status, marital status, educational level, ethnicity and perception of financial security. Statistical analysis consisting of one way analysis of variance, Student Newman-Keuls ad hoc procedure and multiple regression indicated an independent correlation between financial security and education level to life satisfaction scores. Neither ERT nor menopause status was correlated with perceived life satisfaction score of respondents.

[Truncated abstract] Breast cancer is the cause of significant suffering and death in our community. It is now estimated that the risk of developing breast cancer for an Australian woman before the age of 85 is 1 in 8, with this risk rising for unknown reasons. While mortality rates from breast cancer are falling due to increased awareness and early detection, few new treatments have been developed from an advanced understanding of the molecular basis of the disease. From decades of scientific research it is clear that estrogen (E2) has a large role to play in breast cancer. However, the basic mechanism behind E2 action in breast cancer remains unclear. E2 plays a fundamental role in breast cancer cell proliferation and is highly expressed in breast cancers, thus, it is important to understand both E2 and its receptor, the estrogen receptor (ER). The ER is a member of the nuclear receptor (NR) superfamily. The NR superfamily consists of a large group of proteins which regulate a large number of homeostatic proteins together with regulator proteins termed coregulators and corepressors. SRA (steroid receptor RNA activator) is the only known RNA coactivator and augments transactivation by NRs. SRA has been demonstrated to play an important role in mediating E2 action (Lanz et al., 1999; Lanz et al., 2003) and its expression is aberrant in many human breast tumors, suggesting a potential role in breast tumorigenesis (Murphy et al., 2000). Despite evidence that an alternative splice variant of SRA exists as a protein (Chooniedass-Kothari et al., 2004), it has been conclusively shown that SRA can function as an RNA transcript to coactivate NR transcription (Lanz et al., 1999; Lanz et al., 2002; Lanz et al., 2003). The precise mechanism by which SRA augments ER activity remains unknown. However, it is currently hypothesized that SRA acts as an RNA scaffold for other coregulators at the transcription initiation site. Several SRA stem loops have been identified as important for SRA function, including structure (STR) 1, 5 and 7 (Lanz et al., 2002; Zhao et al., 2007). Previously, I sought to identify SRA-binding proteins using a specific stem-loop structure of SRA (STR7) that was identified as both important for its coactivator function (Lanz et al., 2002) and also as a target for proteins from breast cancer cell extracts (Hatchell, 2002). From a yeast E. Hatchell Abstract iii III hybrid screen using STR7 as bait, I identified a novel protein which was named SLIRP (Patent Number: WO/2007/009194): SRA stem-Loop Interacting RNA-binding Protein (Hatchell, 2002; Hatchell et al., 2006). '...' This thesis demonstrates that SLIRP modulates NR transactivation, provides mechanistic insight into interactions between SRA, SRC-1, HSP-60 and NCoR and suggests that SLIRP may regulate mitochondrial function. These studies contribute significantly to the growing field of NR biology, and contribute more specifically to the elucidation of estrogen action in breast cancer. Furthermore, it lays a strong and exciting foundation for further studies to evaluate SLIRP as a biomarker and potential therapeutic target in hormone dependent cancers.

Thesis (PhD)--Stellenbosch University, 2014.<br>ENGLISH ABSTRACT: Phytoestrogen containing extracts of Cyclopia, an indigenous South African fynbos plant used to prepare honeybush tea, may serve as a source of new estrogen analogues. It would be of great benefit if these new analogues would not only prevent the development and progression of breast cancer which, globally, is responsible for the highest number of cancer associated deaths among females, but also have a reduced side-effect profile when compared to current treatments and, in addition, also alleviate menopause associated symptoms. In this study three extracts, P104, SM6Met, and cup-of-tea, from two species of Cyclopia, C. genistoides and C. subternata, were evaluated for their potential to modulate molecular targets involved in prevention and treatment of breast cancer. We show that the phytoestrogenic extracts of Cyclopia antagonise estrogen-induced cell proliferation both in vitro as well as in vivo. Furthermore, our study presents various molecular mechanisms whereby the Cyclopia extracts may be eliciting this effect. Importantly, we show, for the first time, that the Cyclopia extracts behave as ERα antagonists and ERβ agonists which, with respect to the known role of the ER subtypes in breast cancer, where the ERα subtype is associated with the stimulation of cell proliferation and the occurrence of breast cancer, while ERβ ameliorates the action of ERα in breast cancer and could act as an inhibitor of breast cancer development, may be beneficial for the prevention or treatment of breast cancer. In addition, we also show that the extracts of Cyclopia behave as selective estrogen receptor degraders by down-regulating ERα protein levels while stabilising ERβ protein levels, which not only provides a possible molecular explanation for the observed ERα antagonism and ERβ agonism, but, in addition, may be beneficial as higher ERα levels are associated with malignant breast cancer tumours, while higher ERβ levels are associated with benign tumours. Furthermore, we show that the Cyclopia extracts affect the nuclear localization and distribution of both ER subtypes in a manner that provides an additional molecular explanation for the observed ERα antagonism and ERβ agonism. Investigation of the molecular processes involved in the promotion and progression of breast cancer, such as the distribution of cells between the phases of the cell cycle, cancer cell invasion, and the regulation of genes governing these processes provides evidence that the Cyclopia extracts are not as proliferative as estrogen. In addition, Cyclopia extracts display anti-inflammatory properties, which may be beneficial as inflammation is an enabling characteristic in cancer development and progression. Furthermore, this study, for the first time, shows that the phytoestrogenic extracts of Cyclopia are absorbed, are not toxic, and display biological ERα antagonist activity in vivo by retarding uterine growth. Thus, we propose that the Cyclopia extracts act as selective estrogen receptor subtype modulators with potential to be developed as a nutraceutical for the treatment or prevention of breast cancer.<br>AFRIKAANSE OPSOMMING: Fitoëstrogeen-bevattende ekstrakte van Cyclopia, ‘n inheemse Suid Afrikaanse fynbosplant wat gebruik word vir die voorbereiding van heuningbostee, mag as ‘n bron van nuwe estrogeen-analoë dien. Dit sal baie voordelig wees indien hierdie nuwe analoë nie net die ontwikkeling en progressie van borskanker sal voorkom nie, aangesien borskanker wêreldwyd verantwoordelik is vir die grootste getal kankerverwante sterftes onder vroue, maar ook ‘n verminderde newe-effek profiel vertoon in vergelyking met huidige behandelings en ook, boonop, simptome wat met menopouse geassosieer word, sal verlig. In hierdie studie is drie ekstrakte, P104, SM6Met, en cup-of-tea, vanaf twee spesies van Cyclopia, C. genistoides en C. subternata, geëvalueer vir hul potensiaal om die molekulêre teikens betrokke by die voorkoming en behandeling van borskanker te moduleer. Ons wys dat die fitoëstrogeniese ekstrakte van Cyclopia antagoniseer estrogeen-geïnduseerde selproliferasie beide in vitro as ook in vivo. Verder bied ons studie ook verkskeie molekulêre meganismes aan oor hoe die Cyclopia ekstrakte hierdie effek mag ontlok. ‘n Belangrike bevinding is dat ons vir die eerste keer wys dat die Cyclopia ekstrakte hulself as ERα -antagoniste en ERβ-agoniste gedra wat, met betrekking tot die erkende rol van die ER-subtipes in borskanker, waar die ERα-subtipe geassosieer word met die stimulasie van selproliferasie en die gebeurtenis van borskanker, terwyl ERβ die aksie van ERα onderdruk en as ‘n inhibeerder van borskankerontwikkeling kan dien, voordelig mag wees vir die voorkoming of behandeling van borskanker. Ons wys boonop ook dat die ekstrakte van Cyclopia hulself soos selektiewe estrogeen- reseptor-degradeerders gedra deurdat hul ERα-proteïnvlakke verlaag terwyl hul ERβ-proteïnvlakke stabiliseer. Dit verksaf nie net ‘n moontlike molekulêre verduideliking vir die waargeneemde ERα-antagonisme en ERβ-agonisme nie, maar mag ook voordelig wees in borskanker aangesien hoër ERα-vlakke geasosieer word met kwaadaardige borskankertumors en hoër ERβ-vlakke met nie-kwaadaardige tumors. Verder wys ons dat die Cyclopia ekstrakte die lokalisering en verspreiding van beide ER-subtipes in die selkern op so ‘n wyse beïnvloed dat dit ‘n addisionele molekulêre verduideliking bied vir die ERα-antagonisme en ERβ-agonisme wat waargeneem is. Verdere ondersoek van die molekulêre prosesse betrokke by die promosie en progressie van borskanker, soos die verspreiding van selle tussen die fases van die selsiklus, die beweging van kankerselle na omliggende weefsels, en die regulering van gene wat hierdie prosesse beheer, verskaf bewyse dat die Cyclopia-ekstrakte nie so proliferatief is soos estrogeen nie. Die ekstrakte van Cyclopia vertoon boonop ook anti-inflamatoriese eienskappe, wat voordelig mag wees aangesien inflammasie ‘n bydraende eienskap in kankerontwikkeling en -progressie is. Verder wys hierdie studie vir die eerste keer dat die fitoëstrogeniese ekstrakte van Cyclopia geabsorbeer word, nie toksies is nie, en dat hulle biologiese ERα-antagonis aktiwiteit vertoon deurdat hulle uterus-groei vertraag in vivo. Dus stel ons voor dat die Cyclopia-ekstrakte optree soos selektiewe-estrogeen-reseptor-subtipe-moduleerders met die potensiaal om ontwikkel te word as ‘n nutraseutiese middel vir die behandeling of voorkoming van borskanker.

Homocysteine (Hcy) is a sulfur containing amino acid and is a potent neurotoxin. It has been shown that elevated levels of Hcy, termed hyperhomocysteinemia, plays a role in the pathologies of Alzheimer’s disease (AD) and age-related cognitive decline. Hcy is a glutamate agonist, which causes in increase in Ca[superscript (2+)] influx via the activation of NMDA class of excitatory amino acid receptors, which results in neuronal cell death and apoptosis. Estrogen and progesterone are female hormones that are responsible for reproduction and maternal behaviour. However, in the last decade, it is evident that both female hormones have neuroprotective properties in many animal models of neurodegeneration. Collectively, both estrogen and progesterone reduce the consequences of the oxidative stress by enhancing the antioxidant defence mechanisms, reducing excitotoxicity by altering glutamate receptor activity and reducing the damage caused by lipid peroxidation. However, the mechanisms by which estrogen and progesterone provide such neuroprotection probably depend on the type and concentration of hormone present. Moreover, numerous studies have shown that hormone replacement therapy (HRT, estrogen and progestins) or estrogen-only replacement therapy (ERT) may prevent or delay the onset of AD and improve cognition for women with AD. Clinical trials have also shown that women taking HRT may modify the effects of Hcy levels on cognitive functioning. Oxidative stress increases in the aging brain and thus has a powerful effect on enhanced susceptibility to neurodegenerative disease. The detection and measurement of lipid peroxidation and superoxide anion radicals in the brain tissue supports the involvement of free radical reactions in neurotoxicity and in neurodegenerative disorders. The hippocampus is an important region of the brain responsible for the formation of memory. However, agents that induce stress in this area have harmful effects and could lead to dementia. This study aims to investigate and clarify the neuroprotective effects of estrogen and progesterone, using Hcy-induced neurodegenerative models. The initial studies demonstrate that estrogen and progesterone have the ability to scavenge potent free radicals. Histological studies undertaken reveal that both estrogen and progesterone protect against Hcy-induced neuronal cell death. In addition, immunohistochemical investigations show that Hcy-induced apoptosis in the hippocampus can be inhibited by both estrogen and progesterone. However, estrogen also acts at the NMDA receptor as an agonist, while progesterone blocks at the NMDA receptor. These mechanisms reduce the ability of Hcy to cause damage to neurons, since Hcy-induced neurotoxicity is dependent on the overstimulation of the NMDA receptor. SOD and GPx are important enzymatic antioxidants which can react with ROS and neutralize them before these inflict damage in the brain. Hcy can increase oxidative stress by inhibiting expression and function of these antioxidants. However, it has been shown that the antioxidant abilities of both estrogen and progesterone can up-regulate the activities of SOD and GPx. These results provide further evidence that estrogen and progesterone act as antioxidants and are free radical scavengers. The discovery of neuroprotective agents is becoming important as accumulating evidence indicates the protective role of both estrogen and progesterone in Hcy-induced neurodegeneration. Thus further work in clinical trials is needed to examine whether reducing Hcy levels with HRT can become the treatment of neurodegenerative disorders, such as Alzheimer’s disease.

The effects of exogenous estrogen administration on aspects of memory and cognition in women were examined in two studies. In Study 1, women receiving estrogen replacement therapy were compared to untreated women on four measures of verbal memory. Those receiving estrogen had significantly better scores on a measure of delayed memory for propositional material. In Study 2, women in the early stages of Alzheimer's Disease (AD) were administered either estrogen or placebo on a double-blind basis for six months. Women given estrogen showed improvement on a measure of verbal memory and spatial attention compared to the placebo controls. The combined results of these studies provide evidence that estrogen enhances aspects of verbal memory in both healthy postmenopausal women and in postmenopausal women in the early stages of AD as measured by neuropsychological tests. These effects might be mediated by actions of estrogen on neuronal morphology and physiology in brain areas important for memory and cognition.

Memory function was examined in association with sex hormone levels in women. The results of the first study suggest that self-reports of memory problems were especially prevalent among women attending a menopause clinic compared to a nonpatient sample. In the following investigation, women given placebo after undergoing a bilateral oophorectomy showed decreases in memory performance, specifically on a paired-associate learning task, coincident with declines in estrogen levels. Significant improvements were found in estrogen-treated women pre- to postoperatively in the immediate recall of paragraphs, in association with supraphysiological estrogen levels. A final study on naturally-cycling women found a decline in visual memory performance during the menstrual compared to the luteal phase of the cycle. Visual memory scores were positively correlated with progesterone levels whereas paired-associate recall scores were positively associated with estradiol levels during the luteal phase. These results suggest that certain aspects of memory covary with changes in sex steroid levels in some women.

Includes bibliographical references (leaves 254-288). Examines language used by general practitioners and in mass media to ask 'what are the implications of constructions of menopause for health care practice and public health for women at menopause?'. Presents the findings of qualitative analysis of semi-structured interviews with nine general practitioners working in rural South Australia and qualitative and quantitative analyses of 345 south Australian newspaper articles from 1986 to 1998.

OBJETIVOS. Verificar os efeitos da terapia hormonal com derivados estrogênicos e progestogênicos, isolados ou associados à metiltestosterona, na sexualidade e nos sintomas climatéricos em mulheres na pós-menopausa e comparar os dois tipos de terapia hormonal. CASUÍSTICA. Selecionaram-se sessenta mulheres sexualmente ativas, com queixas sexuais, com relacionamento estável com parceiro capacitado para o coito, com idade de 42 a sessenta anos (média etária 52,1 + 4 anos) e tempo de menopausa de um a 28 anos (média 5,6 anos). Excluíram-se mulheres com doenças sistêmicas, doenças psiquiátricas, endócrinas, distopias genitais, tabagistas e usuárias de terapia hormonal ou de medicamentos que apresentavam interferência na sexualidade. METODOLOGIA. Realizou-se estudo de coorte progressiva, duplo-cego, randomizado, com duração de 12 meses. As mulheres foram divididas em dois grupos: EP (n=29), medicadas com estrogênios conjugados (EEC) 0,625 mg + acetato de medroxiprogesterona (AMP) 2,5 mg + placebo, e grupo EP+A (n=31), medicadas com EEC 0,625 mg + AMP 2,5 mg + metiltestosterona 2,0 mg. Para estudar a sexualidade, utilizou-se o Questionário Sexual do Hospital das Clínicas e foram avaliados o desejo sexual, a excitação e a capacidade orgástica nas atividades desenvolvidas com o parceiro, o interesse sexual não vinculado, exclusivamente, às atividades desenvolvidas com o parceiro, a dispareunia, a secura vaginal e a freqüência sexual. Para as análises estatísticas, utilizaram-se Modelos Lineares Gerais para Medidas Repetidas, Análise de Variância (ANOVA), Modelos de Regressão Logística Multinomial e Qui-quadrado de Pearson. O nível de significância foi de 5%. RESULTADOS. Nos grupos EP e EP+A, houve aumento no escore de desejo sexual vinculado, exclusivamente, às atividades desenvolvidas com o parceiro (F=18,334; p<0,001), no escore de excitação sexual (F=14,022; p < 0,001), na capacidade orgástica (F=34,650; p < 0,001) e na freqüência sexual (F=7,687; p=0,008), bem como redução da secura vaginal (?2=44,153; p<0,001), da dispareunia (?2=34,447; p < 0,001) e do índice menopausal de Kupperman (F=158,460; p < 0,001). A análise comparativa entre os grupos EP e EP+A mostrou maior interesse sexual não vinculado, exclusivamente, às atividades com o parceiro (?2=11,551; p=0,021) e mais altos índices de Castelli I (F=8,542; p < 0,001) e índices de Castelli II (F=11,500; p < 0,001) no grupo EP+A. Não se observaram hirsutismo nem alopécia em nenhum dos grupos; acne grau I foi observada em duas mulheres do grupo EP e em 13 do grupo EP+A. CONCLUSÕES. As terapias hormonais com derivados estrogênicos e progestogênicos, isolados ou associados à metiltestosterona, causaram impacto positivo em todos os parâmetros sexuais e nos sintomas climatéricos analisados. A associação de metiltestosterona ao tratamento estro-progestacional aumentou o interesse sexual não vinculado, exclusivamente, às atividades com o parceiro e os índices de Castelli I e II. Nos demais parâmetros estudados, não houve diferença entre os dois grupos<br>Objectives: To verify the effects of hormone therapy with estrogen and progesterone derivatives when used singly or combined with methyltestosterone, on sexuality and on climacteric symptoms in postmenopausal women. Subjects: The series included sixty-sexually active women, with sexual complaints, in a stable relationship with a partner capable of intercourse, ages ranging from 42 to sixty years (average 52,1 + 4) and menopause time from one to 28 years (average 5,6). Excluded were women with systemic diseases, psychiatric and endocrine disorders, genital dystopias, smokers and those on hormone therapy or medications that affect sexuality. Method: A double blind, randomized, progressive cohort study was performed over a twelve month period. The women were divided into two groups: EP (n=29), medicated with conjugated estrogens (EEC) 0,625 mg + medroxyprogesterone acetate (AMP) 2,5 mg + placebo, and group EP+A (n=31), medicated with EEC 0,625 mg + AMP 2,5 mg + methyltestosterone 2,0 mg. For the study of sexuality, the Hospital das Clínicas Sex Questionnaire was utilized, assessing sexual desire linked exclusively to activities developed with the partner; excitation and orgasmic capacity in activities with the partner; sexual interest not linked exclusively to activities developed with the partner; dyspareunia, vaginal dryness and sexual frequency. For statistical analysis, the General Linear Models for Repeated Measures, Analysis of Variance (ANOVA), Multinomial Logistic Regression Models and Pearson Chi square were employed. A 5% significance level was adopted. Results: In groups EP and EP+A, was observed an increase in the sexual desire score linked exclusively to activities developed with the partner (F=18,334; p<0,001), sexual excitation (F=14,002; p<0,001), orgasmic capacity (F=34,650; p < 0,001) and in sexual frequency (F=7,687; p=0,008), as well as an reduction in vaginal dryness (x2=44,153; p < 0,001), dyspareunia (x2=34,447; p < 0,001) and in the Kupperman menopausal index (F=158,460; p < 0,001). Comparative analysis between groups EP and EP+A revealed a greater sexual interest not linked exclusively to activities with the partner (x2=11,551; p=0,021) and higher Castelli I index (F=8,542; p < 0,001) and Castelli II index (F=11,500; p<0,001) in group EP+A. Neither hirsutism nor alopecia were noticed in either group; Class I acne was observed in two women of group EP and in 13 of group EP+A. Conclusion: Hormone therapy with estrogen and progesterone derivatives used singly or together with methyltestosterone had a positive result on all sexual parameters and on climacteric symptoms analyzed. Association of methyltestosterone to estrogen-progesterone treatment increased sexual interest not linked exclusively to activities with the partner and Castelli I and II indexes. No difference between the two groups in the other parameters studied was demonstrated

Christopher John Haines.<br>Thesis (M.D.)--Chinese University of Hong Kong, 1996.<br>Includes bibliographical references (leaves 239-279).<br>LIST OF TABLES --- p.xviii<br>LIST OF FIGURES --- p.xxi<br>LIST OF ABBREVIATIONS --- p.xxii<br>Chapter CHAPTER1 --- INTRODUCTION --- p.1<br>Problems related to the menopause<br>Research plan<br>Chapter CHAPTER2 --- OVERVIEW --- p.15<br>Introduction<br>Atherosclerosis and the lipid profile<br>Coronary artery disease and lipid abnormalities in women<br>"Exogenous oestrogens, progestogens and coronary artery disease "<br>Lipoprotein (a)<br>Chapter CHAPTER3 --- GENERAL METHODOLOGY --- p.134<br>Recruitment of cases<br>Pharmacokinetics<br>Data collection and analysis of samples<br>Ethical considerations<br>Chapter CHAPTER4 --- STUDY I -THE SHORT TERM EFFECTS OF ORAL OESTROGEN --- p.157<br>Crossover analysis of effects of oral oestrogen on lipoprotein (a) and other lipoproteins<br>Relationship between lipoprotein (a) and other lipids and lipoproteins<br>Chapter CHAPTER5 --- STUDY II -THE SUSTAINED EFFECTS OF ORAL OESTROGEN --- p.186<br>Analysis of prolonged effects of oral oestrogen on lipoprotein (a) and other lipids and lipoproteins<br>Chapter CHAPTER6 --- STUDY III -THE EFFECTS OF COMBINED CYCLICAL HORMONE REPLACEMENT THERAPY --- p.196<br>Analysis of effect of combined cyclical hormone replacement therapy on lipoprotein (a) and other lipids and lipoproteins<br>Comparison between sampling during oestrogen alone and combined phase of treatment<br>Chapter CHAPTER7 --- STUDY IV -THE EFFECTS OF PERCUTANEOUS OESTROGEN --- p.214<br>Analysis of effect of percutaneous on lipoprotein (a) and other lipids and lipoproteins<br>Chapter CHAPTER8 --- SUMMARY AND CONCLUSIONS --- p.228<br>BIBLIOGRAPHY --- p.239

by Cheung Tak Chi.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.<br>Includes bibliographical references (leaves 117-123).<br>Abstract also in Chinese.<br>Acknowledgeements --- p.i<br>Abbreviations --- p.ii<br>Abstract --- p.v<br>List of Figures --- p.viii<br>List of Tables --- p.xiv<br>Contents --- p.xv<br>Contents<br>Chapter 1. --- Introduction --- p.1<br>Chapter 1.1 --- Epidemiological Risk Factors --- p.1<br>Chapter 1.1.1 --- Age --- p.1<br>Chapter 1.1.2 --- Race --- p.2<br>Chapter 1.1.3 --- Environmental or Migratory Factor --- p.2<br>Chapter 1.1.4 --- Diet --- p.2<br>Chapter 1.1.5 --- Genetics --- p.3<br>Chapter 1.2 --- Regulation of Normal Prostate Development and Function --- p.4<br>Chapter 1.3 --- Biochemistry and Development of Prostate Cancer --- p.6<br>Chapter 1.3.1 --- Androgen-Dependent Prostate Cancer --- p.6<br>Chapter 1.3.2 --- Androgen-Independent Prostate Cancer --- p.8<br>Chapter 1.4 --- Classification of Prostate Cancer --- p.9<br>Chapter 1.4.1 --- Stage A Prostate Cancer --- p.10<br>Chapter 1.4.2 --- Stage B Prostate Cancer --- p.10<br>Chapter 1.4.3 --- Stage C Prostate Cancer --- p.11<br>Chapter 1.4.4 --- Stage D Prostate Cancer --- p.11<br>Chapter 1.5 --- Methods for Early Detection of Prostate Cancer --- p.12<br>Chapter 1.6 --- Clinical Treatment of Prostate Cancer --- p.12<br>Chapter 1.6.1 --- Surgery --- p.12<br>Chapter 1.6.2 --- Radiotherapy --- p.13<br>Chapter 1.6.3 --- Chemotherapy --- p.13<br>Chapter 1.6.4 --- Hormonal Therapy --- p.13<br>Chapter 1.7 --- Objective --- p.14<br>Chapter 1.8 --- Estrogen and Its Related Compounds --- p.16<br>Chapter 1.8.1 --- 17β-Estradiol --- p.16<br>Chapter 1.8.2 --- Tamoxifen --- p.18<br>Chapter 1.8.3 --- Aromatase Inhibitor --- p.20<br>Chapter 1.9 --- Anticancer Drugs --- p.23<br>Chapter 1.9.1 --- Doxorubicin --- p.23<br>Chapter 1.9.2 --- cis-Platinum --- p.24<br>Chapter 1.10 --- Apoptotic Pathways --- p.25<br>Chapter 1.10.1 --- BCL-2 /BAD Pathway --- p.26<br>Chapter 1.10.2 --- FADD Pathway --- p.27<br>Chapter 1.10.3 --- CAS Pathway --- p.27<br>Chapter 2. --- Materials and Methods --- p.28<br>Chapter 2.1 --- Materials --- p.28<br>Chapter 2.2 --- Cell Lines --- p.32<br>Chapter 2.3 --- Preparation of Drugs --- p.32<br>Chapter 2.4 --- Drug Sensitivity Assay --- p.33<br>Chapter 2.5 --- Cell Cycle Analysis --- p.35<br>Chapter 2.6 --- DNA Fragmentation Assay --- p.36<br>Chapter 2.7 --- Annexin Binding Assay --- p.37<br>Chapter 2.8 --- Western Blot Analysis --- p.38<br>Chapter 2.9 --- Data Analysis --- p.41<br>Chapter 3. --- Results --- p.42<br>Chapter 3.1 --- Response of Human Androgen-Independent Prostate Cancer Cells to Doxorubicin and cis-Platinum --- p.42<br>Chapter 3.2 --- The Effect of 17p-Estradiol on the Growth and Anticancer Drug Sensitivity of Human Androgen-Independent Prostate Cancer Cells --- p.45<br>Chapter 3.2.1 --- 17β-Estradiol on Cell Growth --- p.45<br>Chapter 3.2.2 --- 17β-Estradiol on Anticancer Drug Sensitivity --- p.45<br>Chapter 3.2.3 --- 17β-Estradiol and Doxorubicin on Cell Cycle Progression --- p.51<br>Chapter 3.2.4 --- 17β-Estradiol and Doxorubicin Induced DNA Fragmentation --- p.57<br>Chapter 3.2.5 --- 17β-Estradiol and Doxorubicin on Annexin Staining --- p.59<br>Chapter 3.2.6 --- 17β-Estradiol and Doxorubicin on Apoptotic Protein Expression --- p.62<br>Chapter 3.3 --- The Effect of Tamoxifen on the Growth and Anticancer Drug Sensitivity of Human Androgen-Independent Prostate Cancer Cells --- p.64<br>Chapter 3.3.1 --- Tamoxifen on Cell Growth of Human --- p.65<br>Chapter 3.3.2 --- Tamoxifen on Anticancer Drug Sensitivity --- p.65<br>Chapter 3.3.3 --- Tamoxifen and Doxorubicin on Cell Cycle Progression --- p.71<br>Chapter 3.3.4 --- Tamoxifen and Doxorubicin Induced DNA Fragmentation --- p.76<br>Chapter 3.3.5 --- Tamoxifen and Doxorubicin on Annexin Staining --- p.78<br>Chapter 3.3.6 --- Tamoxifen and Doxorubicin on Apoptotic Protein Expression --- p.79<br>Chapter 3.4 --- The Effect of Aromatase Inhibtiors on the Growth and Anticancer Drug Sensitivity of Human Androgen-Independent Prostate Cancer Cells --- p.81<br>Chapter 3.4.1 --- Aromatase Inhibitors on Cell Growth --- p.81<br>Chapter 3.4.2 --- Aromatase Inhibitors on Anticancer Drug Sensitivity --- p.83<br>Chapter 3.4.3 --- 4-AcA and Doxorubicin on Cell Cycle Progression --- p.93<br>Chapter 3.4.4 --- 4-AcA and Doxorubicin Induced DNA Fragmentation --- p.99<br>Chapter 3.4.5 --- 4-AcA and Doxorubicin on Annexin Staining --- p.100<br>Chapter 3.4.6 --- 4-AcA and Doxorubicin on Apoptotic Protein Expression --- p.102<br>Chapter 4. --- Discussion --- p.105<br>Chapter 4.1 --- 17 β-Estradiol and Anticancer Drug Sensitivity --- p.106<br>Chapter 4.2 --- Tamoxifen and Anticancer Drug Sensitivity --- p.109<br>Chapter 4.3 --- Aromatase Inhibitors and Anticancer Drug Sensitivity --- p.112<br>Chapter 4.4 --- DU145 Cells vs PC3 Cells --- p.115<br>Chapter 5. --- Conclusion and Perspectives --- p.116<br>Chapter 6. --- References --- p.117

A breast cancer cell line stably transfected with the CYP19 gene had been employed for aromatase inhibition. Among the phytochemicals tested, the major dietary flavonoids, such as genistein and daidzein, produced very weak inhibition. On the other hand, the red clover isoflavone biochanin A, the hydroxychalcone butein and the red grape phytoalexin resveratrol were found to be effective aromatase inhibitors. Cell proliferation assay had shown that they could inhibit ER-positive cell proliferation induced by testosterone, and the inhibitory effect was specifically attributed to the reduction of estrogen synthesis. In another breast cancer cell line SK-BR-3, resveratrol, biochanin A and genistein inhibited CYP19 both in enzyme and promoter I.3/II transcriptional levels. The element responsible for the inhibition of aromatase by these phytoestrogens should fall within the region between -556 to -446 by upstream of exon II.<br>Breast cancer is one of the most common cancers affecting women. Estrogen plays an important role in breast cancer initiation and development. The majority of breast tumors are initially dependent upon estrogen to support their growth. Most breast cancers occur in the postmenopausal period. However, the intra-tumoral estradiol (E2) is maintained at a high level equivalent to the pre-menopausal status. High intra-tumoral E2 level in postmenopausal women is sustained by the biosynthesis of estrogens in the tumorous tissue.<br>Genistein and Biochanin A, ranged from 0.1 to 10 muM, might act as estrogen agonist and induced aromatase activity and promoter I.1 transactivation in ERalpha-transfected SK-BR-3 cells. (Abstract shortened by UMI.)<br>The aromatase enzyme, CYP19, belongs to a family of P450 enzyme. As a final rate-limiting step in estrogen biosynthesis, it catalyzes the conversion of C 19 steroids to estrogens. The expression of CYP19 is tissue-specific, and is regulated by alternate promoter usage. The use of aromatase inhibitors for breast cancer treatment has become a major therapeutic approach.<br>The consumption of some phytochemicals protects against breast cancer. Yet the mechanisms are far from clear. In my present study, various phytochemicals, including phytoestrogens, monoterpenes and carotenoids, were evaluated for their effect on aromatase.<br>Wang Yun.<br>"July 2005."<br>Adviser: Lai-Kwok Leung.<br>Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3716.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (p. 145-169).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract in English and Chinese.<br>School code: 1307.

Wong Chi Ming.<br>"August 2004."<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.<br>Includes bibliographical references (p. 182-215).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Mode of access: World Wide Web.<br>Abstracts in English and Chinese.

This investigation was conducted to determine the effect of estrogen replacement therapy (ERT) on vitamin B-6 status of postmenopausal women. Nineteen postmenopausal women served as subjects. Nine (54.7 + 4.7 years) were taking ERT (experimental group); ten (56.8 + 2.3 years) were not (control group). For three consecutive days, subjects recorded their dietary intake and collected their 24-hour urine specimens. On the fourth day, a fasting blood sample was drawn from the subjects. The dietary intake of vitamin B-6, as well as the concentration of total vitamin B-6 in plasma (PB6; and urine (UB6) were measured. PB6 and UB6 were determined by a microbiological method with Saccharomyces uvarum as the assay organism. The mean age, height, hematocrit and hemoglobin values were similar for the two groups. The experimental group was significantly heavier than the control group (p<0.05). The experimental group had a lower mean PB6 than the control group: 47.7 ± 19.7 nmol/L vs. 56.2 + 20.6 nmol/L. These means were not significantly different (p=0.05). PB6 was positively correlated with dietary vitamin B-6 intake (p=0.0001) and vitamin B-6 to protein ratio (p=0.0021). When the means were adjusted for dietary vitamin B-6 and the vitamin B-6 to protein ratio, the mean PB6 of the experimental group (42.7 nmol/L) was significantly lower than that of the control group (60.6 nmol/L) (p<0.05). PB6 was not positively correlated with either age (r=0.20) or the vitamin B-6 dietary history score (r=0.15). UB6 was similar for the two groups. UB6 correlated positively with daily dietary intake of vitamin B-6 (r=0.51, p<0.05) and the ratio of vitamin B-6 to protein (r=0.47, p<0.05), UB6 was not significantly correlated to urine volume (r=0.05). The mean daily intakes of vitamin B-6 and protein were similar for the two groups. One of the 19 subjects had a vitamin B-6 intake that was less than 67 percent of the RDA. Most subjects' (89%) intake of vitamin B-6 was adequate when the ratio of 0.016 mg of vitamin B-6 per g of protein was used as the standard.<br>Graduation date: 1990

by Chow Ka Yee.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.<br>Includes bibliographical references (leaves 203-221).<br>Abstracts in English and Chinese.<br>Acknowledgements --- p.i<br>Abstract --- p.ii<br>Abstract in Chinese --- p.iv<br>List of Abbreviations --- p.vi<br>Table of Contents --- p.xi<br>List of Figures --- p.xviii<br>List of Tables --- p.xxii<br>Chapter CHAPTER 1 --- INTRODUCTION --- p.1<br>Chapter 1.1 --- The Characteristics of Arsenic Trioxide (AS2O3) --- p.2<br>Chapter 1.2 --- The Therapeutic Applications of Arsenic Trioxide (As203) --- p.5<br>Chapter 1.3 --- Acute Promyelocytic Leukemia (APL) --- p.6<br>Chapter 1.3.1 --- Pathologies of APL --- p.7<br>Chapter 1.3.2 --- All Trans Retinoic Acid (ATRA) Treatment of APL Patients --- p.7<br>Chapter 1.3.3 --- Clinical Trials of Arsenic Trioxide (As203) on APL Patients --- p.9<br>Chapter 1.3.4 --- In Vitro and In Vivo Studies of Arsenic Trioxide (As203) in the Treatment of APL --- p.10<br>Chapter 1.3.4.1 --- Induction of Apoptosis --- p.11<br>Chapter 1.3.4.2 --- Induction of Cell Differentiation --- p.11<br>Chapter 1.3.5 --- General Toxicity and Side Effects of Arsenic Trioxide (AS2O3) on APL Patients --- p.12<br>Chapter 1.4 --- Effects of Arsenic Trioxide (As203) on Other Primary Cancer Cells and Cancer Cell Lines --- p.12<br>Chapter 1.5 --- Epidemiology of Breast Cancer --- p.14<br>Chapter 1.6 --- Classification of Breast Cancer --- p.17<br>Chapter 1.7 --- Etiology of Breast Cancer --- p.17<br>Chapter 1.8 --- Hormones and Breast Cancer --- p.18<br>Chapter 1.9 --- Estrogen Receptors (ER) --- p.20<br>Chapter 1.9.1 --- Structures of Estrogen Receptors (ER) --- p.21<br>Chapter 1.9.2 --- Estrogen Receptors (ER) Mediated Signaling Pathway --- p.22<br>Chapter 1.9.2.1 --- Ligand Dependent Pathway --- p.22<br>Chapter 1.9.2.2 --- Ligand Independent Pathway --- p.22<br>Chapter 1.9.2.3 --- Estrogen Response Element (ERE)-Independent Pathway --- p.23<br>Chapter 1.9.2.4 --- Non-Genomic Pathway --- p.23<br>Chapter 1.9.3 --- Estrogen Receptors (ER) Regulated Gene Expression --- p.25<br>Chapter 1.10 --- Current Therapy of Breast Cancer --- p.26<br>Chapter 1.10.1 --- Hormonal Therapy (Anti-Estrogenicity) --- p.26<br>Chapter 1.10.1.1 --- Tamoxifen --- p.26<br>Chapter 1.10.1.2 --- Other Pure Anti-Estrogens --- p.28<br>Chapter 1.10.2 --- Regulation of Estrogen Receptors (ER) and Transcription Coregulators --- p.29<br>Chapter 1.10.3 --- Apoptosis Induction --- p.29<br>Chapter 1.11 --- Aims of Study --- p.30<br>Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.32<br>Chapter 2.1 --- Materials --- p.33<br>Chapter 2.1.1 --- Cell Lines and Culture Media --- p.33<br>Chapter 2.1.1.1 --- Cell Lines --- p.33<br>Chapter 2.1.1.2 --- Culture Media --- p.34<br>Chapter 2.1.2 --- Chemicals --- p.35<br>Chapter 2.1.3 --- Reagents and Buffers --- p.36<br>Chapter 2.1.3.1 --- Reagents for MTT Assay --- p.36<br>Chapter 2.1.3.2 --- Reagents for [methyl-3H] Thymidine Incorporation into DNA --- p.37<br>Chapter 2.1.3.3 --- Reagents for Trypan Blue Exclusion Assay --- p.37<br>Chapter 2.1.3.4 --- Reagents and Buffers for Western Blot Analysis --- p.37<br>Chapter 2.1.3.5 --- Reagents and Buffers for Flow Cytometry --- p.40<br>Chapter 2.1.3.6 --- Reagents and Buffers Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.40<br>Chapter 2.1.3.7 --- Reagents for Transfection and Luciferase Reporter Assay --- p.41<br>Chapter 2.1.3.8 --- Reagents and Buffers for In Vivo Studies --- p.42<br>Chapter 2.2 --- Methods --- p.42<br>Chapter 2.2.1 --- In Vitro Studies --- p.42<br>Chapter 2.2.1.1 --- Cell Treatment --- p.42<br>Chapter 2.2.1.2 --- Drug Preparation --- p.43<br>Chapter 2.2.1.3 --- MTT Assay --- p.43<br>Chapter 2.2.14 --- Trypan Blue Exclusion Assay --- p.44<br>Chapter 2.2.1.5 --- [methyl-3H] Thymidine Incorporation into DNA --- p.45<br>Chapter 2.2.1.6 --- Detection of DNA Fragmentation --- p.45<br>Chapter 2.2.1.7 --- ERα Competitive Binding Assay --- p.47<br>Chapter 2.2.1.8 --- Cell Cycle Analysis by Flow Cytometry with Propidium Iodide (PI) Staining --- p.48<br>Chapter 2.2.1.9 --- Cell Cycle Analysis by Flow Cytometry with Annexin V-PI Staining --- p.48<br>Chapter 2.2.1.10 --- Cell Cycle Analysis by Flow Cytometry with JC-1 Staining --- p.49<br>Chapter 2.2.1.11 --- Cell Cycle Analysis by Flow Cytometry with Hydroethidine (HE) Staining --- p.50<br>Chapter 2.2.1.12 --- Western Blot Analysis of Proteins --- p.50<br>Chapter 2.2.1.13 --- Assessment of the Transcriptional Activity of ERα --- p.55<br>Chapter 3.2.1.14 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.57<br>Chapter 2.2.2 --- In Vivo Studies --- p.61<br>Chapter 2.2.2.1 --- Animal Models --- p.61<br>Chapter 2.2.2.2 --- Treatment Schedules --- p.61<br>Chapter 2.2.2.3 --- Sacrifice of Nude Mice --- p.61<br>Chapter 2.2.2.4 --- Enzymatic Assays --- p.62<br>Chapter 2.2.2.4.1 --- Aspartate Transaminase (AST) --- p.63<br>Chapter 2.2.2.4.2 --- Alanine Transaminase (ALT) --- p.64<br>Chapter 2.2.2.4.3 --- Creatine Kinase (CK) --- p.65<br>Chapter 2.2.2.4.4 --- Lactate Dehydrogenase (LDH) --- p.66<br>Chapter CHAPTER 3 --- "Effects of Arsenic Trioxide (As203) on Human Breast Adenocarcinoma Cell Line, MCF-7 Cell Line" --- p.68<br>Chapter 3.1 --- Introduction --- p.69<br>Chapter 3.2 --- Effect of As203 on Cell Survival of MCF-7 cells by MTT Assay --- p.70<br>Chapter 3.3 --- Cytotoxicity of As203 on MCF-7 Cells by Trypan Blue Exclusion Assay --- p.72<br>Chapter 3.4 --- Effect of As203 on DNA Synthesis and Cell Proliferation of MCF-7 cells by [methyl-3H] Thymidine Incorporation into DNA --- p.76<br>Chapter 3.5 --- Comparison of Cytotoxicity of AS2O3 on MCF-7 Cells with that of Tamoxifen --- p.79<br>Chapter 3.6 --- Summary --- p.82<br>Chapter CHAPTER 4 --- Effects of Arsenic Trioxide (As203) on 17β Estradiol Stimulated MCF-7 cells --- p.83<br>Chapter 4.1 --- Introduction --- p.84<br>Chapter 4.2 --- Effect of 17β estradiol on Cell Viability of MCF-7 Cells by MTT Assay --- p.86<br>Chapter 4.3 --- Effect of As203 and 17β Estradiol on Cell Survival of MCF-7 Cells by MTT Assay --- p.88<br>Chapter 4.4 --- Cytotoxicity of As203 on 17β Estradiol Stimulated MCF-7 cells by Cell Number Counting with Hemacytometer --- p.92<br>Chapter 4.5 --- Growth Inhibitory Effect of As203 on 17β Estradiol stimulated MCF-7 cells by [methyl-3H] Thymidine Incorporation into DNA --- p.94<br>Chapter 4.6 --- "Effect of As203 on Cell Survival of Hormone Independent Breast Cancer Cell Line, MDA-MB-231 Cells" --- p.96<br>Chapter 4.7 --- Summary --- p.100<br>Chapter CHAPTER 5 --- Effects of Arsenic Trioxide (As203) on Normal Cells --- p.102<br>Chapter 5.1 --- Introduction --- p.103<br>Chapter 5.2 --- "Effect of As203 on Normal Human Fibroblast Cell Line, Hs68" --- p.104<br>Chapter 5.3 --- Effects of As203 on the Normal Cells of Nude Mice --- p.106<br>Chapter 5.3.1 --- Effect of AS2O3 on Aspartate Transaminase (AST) Activity of Nude Mice --- p.107<br>Chapter 5.3.2 --- Effect of As203 on Alanine Transaminase (ALT) Activity of Nude Mice --- p.109<br>Chapter 5.3.3 --- Effect of As203 on Creatine Kinase (CK) Activity of Nude Mice TABLE OF CONTENTS --- p.111<br>Chapter 5.3.4 --- Effect of As203 on Lactate Dehydrogenase (LDH) Activity of Nude Mice --- p.113<br>Chapter 5.4 --- Summary --- p.115<br>Chapter CHAPTER 6 --- Action Mechanisms underlying the Survival Inhibitory Effects of Arsenic Trioxide (As203) on MCF-7 cells --- p.116<br>Chapter 6.1 --- Introduction --- p.117<br>Chapter 6.2 --- Detection of Apoptosis --- p.119<br>Chapter 6.2.1 --- Detection of DNA Fragmentation --- p.119<br>Chapter 6.2.2 --- Phosphatidylserine (PS) Externalization Detected by Flow Cytometry with Annexin V-PI Staining --- p.124<br>Chapter 6.2.2.1 --- The Principle --- p.124<br>Chapter 6.2.2.2 --- PS Externalization upon AS2O3 Treatment --- p.126<br>Chapter 6.3 --- Analysis of Cell Cycle Distribution of MCF-7 Cells --- p.130<br>Chapter 6.3.1 --- The Principle --- p.130<br>Chapter 6.3.2 --- Regulation of Cell Cycle Distribution of MCF-7 Cells upon As2O3 Treatment --- p.131<br>Chapter 6.4 --- The Action Mechanisms Underlying As203 Induced Apoptosis or Cell Cycle Arrest --- p.137<br>Chapter 6.4.1 --- Effect of As203 on Mitochondrial Membrane Potential of MCF-7 Cells --- p.137<br>Chapter 6.4.2 --- Regulation of Free Oxidative Species (ROS) Production in MCF-7 Cells upon AS2O3 Treatment --- p.140<br>Chapter 6.4.2.1 --- Analysis of Superoxide Production in MCF-7 Cells upon AS2O3 Treatment by Flow Cytometry with Hydroethidine (HE) Staining --- p.140<br>Chapter 6.4.2.2 --- Effect of As203 on Cell Survival of MCF-7 Cells Co-treated with N-Acteyl-L-Cysteine (NAC) by MTT Assay --- p.143<br>Chapter 6.4.3 --- Regulation of Bcl-2 Protein Level in MCF-7 Cells upon As2O3 Treatment --- p.145<br>Chapter 6.4.4 --- Regulation of p53 Protein Level in MCF-7 Cells upon AS2O3 Treatment --- p.147<br>Chapter 6.5 --- Summary --- p.149<br>Chapter CHAPTER 7 --- Effects of Arsenic Trioxide (As203) on Estrogen Receptor a (ERα) Mediated Signaling Pathway in MCF-7 cells --- p.150<br>Chapter 7.1 --- Introduction --- p.151<br>Chapter 7.2 --- Effect of As203 on Estrogen Binding to Estrogen Receptor a (ERα) by ERα Competitive Binding Assay --- p.152<br>Chapter 7.3 --- Regulation of Estrogen Receptor a (ERα) mRNA Level upon As2O3 Treatment by RT-PCR --- p.156<br>Chapter 7.4 --- Regulation of Estrogen Receptor a (ERα) Protein Level upon As2O3 Treatment --- p.159<br>Chapter 7.5 --- Regulation of Estrogen Receptor a (ERα) Transcriptional Activity upon AS2O3 treatment --- p.161<br>Chapter 7.6 --- "Regulation of Estrogen Target Gene, c-myc, Protein Level upon As2O3 Treatment" --- p.164<br>Chapter 7.7 --- Effects of As203 on Cell Cycle Distribution of MCF-7 Cells under Estrogens Stimulation --- p.167<br>Chapter 7.8 --- Summary --- p.173<br>Chapter CHAPTER 8 --- Discussion --- p.174<br>Chapter 8.1 --- The Anti-Tumor Effects of As203 on MCF-7 Cells --- p.175<br>Chapter 8.2 --- Cytotoxicity of As203 on MCF-7 Cells --- p.175<br>Chapter 8.2.1 --- Induction of Apoptosis in MCF-7 Cells upon As2〇3 Treatment --- p.176<br>Chapter 8.2.2 --- Action Mechanisms Underlying the Induction of Apoptosis by As2〇3 --- p.178<br>Chapter 8.3 --- Growth Inhibition of As203 on MCF-7 Cells --- p.182<br>Chapter 8.3.1 --- Cell Cycle Regulation of MCF-7 Cells upon As203 Treatment --- p.182<br>Chapter 8.4 --- Growth Inhibitory Effects of As203 on Estrogen Stimulated MCF-7 Cells --- p.186<br>Chapter 8.4.1 --- Regulation of Estrogen Receptor a (ERα) Signaling Pathway in MCF-7 cells upon as2o3 Treatment --- p.188<br>Chapter 8.5 --- Cross Talk of ERα Signaling Pathway and Apoptosis in Mediating the Anti-Tumor Effects of As203 on MCF-7 Cells --- p.195<br>Chapter 8.6 --- Toxicity of AS2O3 towards Normal Tissues --- p.197<br>Chapter CHAPTER 9 --- Conclusion and Future Perspectives --- p.200<br>Chapter 9.1 --- Conclusion --- p.200<br>Chapter 9.2 --- Future Perspectives --- p.202<br>References --- p.203

Leung Hok Sum.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (leaves 128-147).<br>Abstracts in English and Chinese.<br>Declaration --- p.i<br>Acknowledgements --- p.ii<br>Abbreviation --- p.iii<br>Abstract in English --- p.iv<br>Abstract in Chinese --- p.vi<br>Contents --- p.viii<br>Chapter Chapter I - --- Introduction<br>Chapter 1.1. --- Steroid Hormone --- p.1<br>Chapter 1.2. --- Estrogen Receptors --- p.2<br>Chapter 1.3. --- Selective Estrogen Receptor Modulators --- p.5<br>Chapter 1.3.1. --- Tamoxifen --- p.5<br>Chapter 1.3.1.1. --- Cardiovascular Effects of Tamoxifen --- p.6<br>Chapter 1.3.1.2. --- Acute Vascular Effects of Tamoxifen --- p.6<br>Chapter 1.3.1.3. --- Chronic Vascular Effects of Tamoxifen --- p.7<br>Chapter 1.3.1.4. --- Antioxidant Effects of Tamoxifen --- p.8<br>Chapter 1.3.2. --- Raloxifene --- p.8<br>Chapter 1.3.2.1. --- Cardiovascular Effects of Raloxifene --- p.8<br>Chapter 1.3.2.2. --- Acute Vascular Effects of Raloxifene --- p.9<br>Chapter 1.3.2.3. --- Chronic Vascular Effects of Raloxifene --- p.10<br>Chapter 1.3.2.4. --- Ovariectomy and Raloxifene Treatment --- p.11<br>Chapter 1.4. --- Mechanism of Action of SERMs --- p.15<br>Chapter 1.5. --- Effects of Functional Endothelium and Nitric Oxide --- p.18<br>Chapter 1.6. --- Dihydropyridine (DHP) Calcium Channel Antagonists --- p.19<br>Chapter 1.6.1. --- Development of Newer Generation of Dihydropyridines --- p.19<br>Chapter 1.6.2. --- Effects of Dihydropyridines on Vascular Endothelium (I) --- p.20<br>Chapter 1.6.3. --- Effects of Dihydropyridines on Vascular Endothelium (II) --- p.21<br>Chapter 1.6.4. --- Effects of Dihydropyridines on Nitric Oxide Synthase (NOS) --- p.21<br>Chapter 1.6.5. --- Clinical Studies of Dihydropyridines --- p.22<br>Chapter 1.7. --- Vascular Ion Channels --- p.25<br>Chapter 1.8. --- Objectives of The Present Study --- p.26<br>Chapter Chapter II - --- Materials and Methods<br>Chapter 2.1. --- Tissue Preparation --- p.27<br>Chapter 2.1.1. --- Preparation of The Porcine Left Circumflex Coronary Arteries --- p.27<br>Chapter 2.1.2. --- Removal of Functional Endothelium --- p.27<br>Chapter 2.1.3. --- Organ Bath Setup --- p.27<br>Chapter 2.1.4. --- Isometric Force Measurement --- p.29<br>Chapter 2.2. --- In situ Endothelial [Ca2+]i Imaging --- p.29<br>Chapter 2.2.1. --- Preparation of Porcine Left Circumflex Coronary Arteries --- p.29<br>Chapter 2.2.2. --- Setup For In situ Endothelial [Ca2+]i Imaging --- p.30<br>Chapter 2.3. --- Electrophysiological Measurement of BKCa Current --- p.31<br>Chapter 2.3.1. --- Enzymatic Dissociation of Coronary Artery Smooth Muscle Cells --- p.31<br>Chapter 2.3.2. --- Electrophysiological Measurement --- p.31<br>Chapter 2.4. --- DPPH Free Radical Scavenging Assay --- p.31<br>Chapter 2.5. --- Solutions and Drugs --- p.32<br>Chapter 2.5.1. --- "Drugs, Chemicals and Enzymes" --- p.32<br>Chapter 2.5.2. --- Solutions Used in Force Measurement --- p.34<br>Chapter 2.6. --- Statistical Analysis --- p.34<br>Chapter Chapter III - --- Tamoxifen-Induced Endothelial Nitric Oxide-Dependent Relaxation in Porcine Coronary Arteries via Ouabain- and BaCl2-Sensitive Mechanisms<br>Chapter 3.1. --- Abstract --- p.35<br>Chapter 3.2. --- Introduction --- p.36<br>Chapter 3.3. --- Methods and Materials --- p.37<br>Chapter 3.3.1. --- Vessel Preparation --- p.37<br>Chapter 3.3.2. --- Isometric Force Measurement --- p.38<br>Chapter 3.3.3. --- In situ Endothelial [Ca2+]i Imaging --- p.39<br>Chapter 3.3.4. --- Chemicals --- p.40<br>Chapter 3.3.5. --- Data Analysis --- p.40<br>Chapter 3.4. --- Results --- p.41<br>Chapter 3.4.1. --- Relaxant Responses --- p.41<br>Chapter 3.4.2. --- Effects of Inhibitors of NO-Dependent Relaxation --- p.41<br>Chapter 3.4.3. --- Effects of Putative K+ Channel Blockers and Ouabain --- p.41<br>Chapter 3.4.4. --- "Effects of Ouabain, Removal of Extracellular K+ Ions and BaCI2" --- p.42<br>Chapter 3.4.5. --- SNP-Induced Relaxation --- p.42<br>Chapter 3.4.6. --- Effects of Actinomycin D and Cycloheximide --- p.42<br>Chapter 3.4.7. --- Relaxant Effect of 17β-Estradiol --- p.43<br>Chapter 3.4.8. --- Effects on Endothelial [Ca2+]i in Isolated Coronary Arteries With Endothelium --- p.43<br>Chapter 3.5. --- Discussion --- p.53<br>Chapter Chapter IV - --- Endothelium-Independent Relaxation to Raloxifene in Porcine Coronary Arteries<br>Chapter 4.1. --- Abstract --- p.57<br>Chapter 4.2. --- Introduction --- p.58<br>Chapter 4.3. --- Methods and Materials --- p.59<br>Chapter 4.3.1. --- Vessel Preparation --- p.59<br>Chapter 4.3.2. --- Isometric Force Measurement --- p.60<br>Chapter 4.3.3. --- Electrophysiological Measurement of BKCa Current --- p.61<br>Chapter 4.3.3.1. --- Enzymatic Dissociation of Coronary Artery Smooth Muscle --- p.61<br>Chapter 4.3.3.2. --- Electrophysiological Measurement --- p.62<br>Chapter 4.3.4. --- Chemicals --- p.63<br>Chapter 4.3.5. --- Data Analysis --- p.63<br>Chapter 4.4. --- Results --- p.64<br>Chapter 4.4.1. --- Effect of Raloxifene on Agonist-Induced Contractions --- p.64<br>Chapter 4.4.2. --- Role of Endothelium --- p.64<br>Chapter 4.4.3. --- Effect of ER Antagonist --- p.65<br>Chapter 4.4.4. --- Effect of Putative K+ Channel Blockers --- p.65<br>Chapter 4.4.5. --- Effect of Elevated Extracellular K+ Concentrations --- p.65<br>Chapter 4.4.6. --- Effects of Raloxifene on BKCa Current --- p.65<br>Chapter 4.5. --- Discussion --- p.75<br>Chapter Chapter V - --- Therapeutic Concentrations of Raloxifene Augment Bradykinin Mediated Nitric Oxide-Dependent Relaxation in Porcine Coronary Arteries<br>Chapter 5.1. --- Abstract --- p.78<br>Chapter 5.2. --- Introduction --- p.79<br>Chapter 5.3. --- Methods and Materials --- p.80<br>Chapter 5.3.1. --- Vessel Preparation --- p.80<br>Chapter 5.3.2. --- Isometric Force Measurement --- p.80<br>Chapter 5.3.3. --- In situ Endothelial [Ca2+]i Imaging --- p.81<br>Chapter 5.3.4. --- Free Radical Scavenging Assay --- p.82<br>Chapter 5.3.5. --- Chemicals --- p.83<br>Chapter 5.3.6. --- Data Analysis --- p.83<br>Chapter 5.4. --- Results --- p.84<br>Chapter 5.4.1. --- Relaxation to Bradykinin --- p.84<br>Chapter 5.4.2. --- Effect of Raloxifene on Bradykinin-Induced Relaxation --- p.84<br>Chapter 5.4.3. --- Effect of Raloxifene on Relaxation Induced by Substance P and --- p.85<br>Chapter 5.4.4. --- Effect of Estrogen on Bradykinin-Induced Relaxation --- p.85<br>Chapter 5.4.5. --- Effect of Raloxifene on Sodium Nitroprusside-Induced Relaxation --- p.86<br>Chapter 5.4.6. --- Free Radical Scavenging Effect --- p.86<br>Chapter 5.4.7. --- Raloxifene Augmentation of Bradykinin-Stimulated Endothelial [Ca2+]i --- p.86<br>Chapter 5.5. --- Discussion --- p.99<br>Chapter Chapter VI - --- "Cilnidipine, a Slow-Acting Ca2+ Channel Blocker, Induces Relaxation in Porcine Coronary Arteries: Role of Endothelial Nitric Oxide and [Ca2+]i"<br>Chapter 6.1. --- Abstract --- p.102<br>Chapter 6.2. --- Introduction --- p.103<br>Chapter 6.3. --- Methods and Materials --- p.104<br>Chapter 6.3.1. --- Vessel Preparation --- p.104<br>Chapter 6.3.2. --- Isometric Force Measurement --- p.105<br>Chapter 6.3.3. --- In situ Endothelial [Ca2+]i Imaging --- p.106<br>Chapter 6.3.4. --- Free Radical Scavenging Assay --- p.107<br>Chapter 6.3.5. --- Chemicals --- p.108<br>Chapter 6.3.6 --- Data Analysis --- p.108<br>Chapter 6.4. --- Results --- p.108<br>Chapter 6.4.1. --- Relaxant Responses --- p.108<br>Chapter 6.4.2. --- Role of the Endothelium --- p.109<br>Chapter 6.4.3. --- Effect of Inhibitors of NO-Dependent Relaxation --- p.109<br>Chapter 6.4.4. --- Effect of Indomethacin and w-conotoxin --- p.110<br>Chapter 6.4.5. --- Effect of Cilnidipine on Sodium Nitroprusside-Induced Relaxation --- p.110<br>Chapter 6.4.6. --- Effects on Endothelial [Ca2+]i in Isolated Endothelium-Intact Coronary Arteries --- p.110<br>Chapter 6.4.7. --- Free Radical Scavenging Effect --- p.110<br>Chapter 6.5. --- Discussion --- p.120<br>Chapter Chapter VII - --- General Summary --- p.123<br>References --- p.128

Yuen Yee Man.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (leaves 108-125).<br>Abstracts in English and Chinese.<br>TITLE PAGE --- p.1<br>ACKNOWLEGDEMENTS --- p.2<br>ABSTRACT --- p.3<br>摘要 --- p.5<br>table of contents --- p.7<br>list of figures and tables --- p.13<br>CHAPTER 1 GENERAL INTRODUCTION --- p.16<br>Chapter 1.1 --- role of PHYTOESTROGENS in soy and red WINE the PREVENTION OF CARDIOVASCULAR DISEASES (CVD) --- p.17<br>Chapter 1.1.1 --- INTRoduction and Classification of Phytoestrogens --- p.17<br>Chapter 1.1.2 --- estrogenic1ty of phytoestrogens and theIr abundancesin Plasma --- p.18<br>Chapter 1.1.3 --- phytoestrogens as one of the active components In cvd Protection --- p.21<br>Chapter 1.1.4 --- effects of Phytoestrogens on LDL Receptor and Apolipoprotein A-1 --- p.22<br>Chapter 1.2 --- role of estrogen receptors (ers) in gene regulation --- p.24<br>Chapter 1.2.1 --- "structure, Classification and tissue distribution of ERS" --- p.24<br>Chapter 1.2.2 --- ligands for ERS --- p.25<br>Chapter 1.2.3 --- mechaniSMS OF LIgands-ERS complex in GENE regulation --- p.27<br>Chapter 1.2.4 --- ligand-independent ER activation --- p.28<br>Chapter 1.3 --- aims and scopes of investigation --- p.29<br>Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.30<br>Chapter 2.1 --- chemicals and materials --- p.30<br>Chapter 2.1.1 --- Chemicals --- p.30<br>Chapter 2.1.2 --- Plasmids --- p.30<br>Chapter 2.2 --- mammalian cell culture maintainence --- p.30<br>Chapter 2.2.1 --- Maintenance of Cells --- p.31<br>Chapter 2.2.2 --- Preparation of Cell Stock --- p.31<br>Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.31<br>Chapter 2.3 --- manipulation of dna --- p.31<br>Chapter 2.3.1 --- isolation of HEPG2 cells genonmic DNA --- p.31<br>Chapter 2.3.2 --- separation and purification of dna from agarose gel --- p.31<br>Chapter 2.3.3 --- Restriction digestionof DNA --- p.32<br>Chapter 2.3.4 --- Ligation of DNA Fragments --- p.32<br>Chapter 2.3.5 --- Transformation of --- p.32<br>Chapter 2.3.6 --- Small Scale Plasmids Purification from DH5a --- p.32<br>Chapter 2.4 --- construction of expression and reporter plasmids --- p.33<br>Chapter 2.4.1 --- Construction of Estrogen Receptorα (Erα) Expression Vectors --- p.33<br>Chapter 2.4.2 --- construction of reporter vectors of LDLR promoter and the Respective Mutants --- p.33<br>Chapter 2.4.3 --- Construction of Reporter Vectors of APOAI Promoter and the Respective Mutants --- p.33<br>Chapter 2.5 --- determination of promoter transcrtiption activities --- p.34<br>Chapter 2.5.1 --- Transient Transfection of Cell with ERa Expression Vector and Promoter Reporter using Lipofectamine PLUS Reagent --- p.34<br>Chapter 2.5.2 --- Dual Luciferase Assay --- p.34<br>Chapter 2.6 --- semi-quantitative and quantitative rt-pcr assay --- p.34<br>Chapter 2.6.1 --- Transient transfection of Cell with ERa Expression Vector Using Lipofectamine PLUS Reagent --- p.34<br>Chapter 2.6.2 --- "Isolation of RNA using TRIzol® Reagent (Life Technology, USA)" --- p.35<br>Chapter 2.6.3 --- Quantitation of RNA --- p.35<br>Chapter 2.6.4 --- First Strand cDNA Synthesis --- p.35<br>Chapter 2.6.5 --- Sem卜Quantitative PCR Reactions --- p.35<br>Chapter 2.6.6 --- Quantitative PCR Reactions --- p.36<br>Chapter 2.7 --- western blotting analysis --- p.36<br>Chapter 2.8 --- statistical methods --- p.36<br>Chapter CHAPTER 3 --- REGULATION BY PHYSIOLOGICAL LEVEL OF 17B-ESTRADIOL ON APOLIPOPROTEIN A-I AND LOW-DENSITY- LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.37<br>Chapter 3.1 --- introduction --- p.37<br>Chapter 3.2 --- results --- p.39<br>Chapter 3.2.1 --- Determination of transient transfection functionality of estrogen receptors in hepg2 cells --- p.39<br>Chapter 3.2.2 --- Effect of 17β-Estradiolon LDLR promoter transcription activity --- p.39<br>Chapter 3.2.3 --- Effect of 17β-Estradiol on apoai promoter transcription activity --- p.40<br>Chapter 3.2 --- discussion --- p.47<br>Chapter CHAPTER 4 --- SOY ISOFLAVONES AND RESVERATROL DISPLAY DIFFERENT MECHANISM IN THE UP-REGULATION OF LOVV-DENSITY-LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.49<br>Chapter 4.1 --- introduction --- p.49<br>Chapter 4.2 --- results --- p.54<br>Chapter 4.2.1 --- Association of ERα and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54<br>Chapter 4.2.2 --- Association of ERβ and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54<br>Chapter 4.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol induced LDLR promoter transcription" --- p.55<br>Chapter 4.2.4 --- Identification of promoter regions responsible for induction of LDLR transcription by isoflavones in the presence OF ERα --- p.55<br>Chapter 4.2.5 --- Identification of promoter regions responsible for induction of LDLR TRANSCRIPTION BY resveratrol IN THE ABSENCE OF ERα --- p.56<br>Chapter 4.3 --- DISCUSSION --- p.75<br>Chapter CHAPTER 5 --- SOY ISOFLAVONES AND RESVERATROL UP-REGULATE APOLIPOPROTEIN A-I SIMILAR TO 17B-ESTRADIOL IN HEPG2 CELLS --- p.80<br>Chapter 5.1 --- INTRODUCTION --- p.80<br>Chapter 5.2 --- RESULTS --- p.84<br>Chapter 5.2.1 --- Association of ERα phytoestrogens on APCAI gene expression --- p.84<br>Chapter 5.2.2 --- Association of ERβ and isoflavones or resveratrol on APOAI promoter transcription activity --- p.85<br>Chapter 5.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol in APOAI promoter transcription in the presence of ERα" --- p.85<br>Chapter 5.2.4 --- Identification of promoter regions responsible for induction of APOAI transcription by isoflavones and resveratrol in the presence of ERα --- p.85<br>Chapter 5.3 --- DISCUSSION --- p.100<br>Chapter CHAPTER 6 --- GENERAL DISCUSSION --- p.103<br>Chapter CHAPTER 7 --- SUMMARY --- p.106<br>BIBLIOGRAPHY --- p.108<br>APPENDIX 1 ABBREVIATIONS --- p.126<br>APPENDIX 2 MATERIALS AND METHODS --- p.129<br>APPENDIX 3 PRIMER LISTS --- p.145<br>APPENDIX 4 REAGENTS AND BUFFERS --- p.147

Po Lai See.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.<br>Includes bibliographical references (leaves 142-152).<br>Abstracts in English and Chinese.<br>TITLE PAGE --- p.p.1<br>ACKNOWLEGDEMENTS --- p.p.2<br>ABSTRACT --- p.p.3<br>摘要 --- p.p.6<br>TABLE OF CONTENTS --- p.p.9<br>LIST OF FIGURES AND TABLES --- p.p.16<br>Chapter CHAPTER 1 --- GENERAL INTRODUCTION<br>Chapter 1.1 --- Estrogen and Estrogen Receptors and its Action --- p.p.18<br>Chapter 1.1.1 --- Estrogen --- p.p.19<br>Chapter 1.1.2 --- Estrogen Receptors --- p.p.19<br>Chapter 1.1.3 --- Structural Differences between ERa and ERp --- p.p.21<br>Chapter 1.1.4 --- Functional Differences --- p.p.22<br>Chapter 1.1.5 --- Effects of Selective Estrogen Receptor Modulators --- p.p.22<br>Chapter 1.1.6 --- Estrogen works --- p.p.23<br>Chapter 1.1.7 --- Estrogen Receptors and Breast Cancer --- p.p.24<br>Chapter 1.2 --- Flavonoids： Properties and Biological Activities --- p.p.25<br>Chapter 1.2.1 --- Chemical Structure and Classification of flavonoids --- p.p.25<br>Chapter 1.2.2 --- Biological Properties and Action Mechanism of Flavonoids… --- p.p.27<br>Chapter 1.2.3 --- Flavonoids and breast cancer prevention --- p.p.27<br>Chapter 1.3 --- Aims and Scopes of Investigation --- p.p.29<br>Chapter CHAPTER 2 --- MATERIALS AND METHODS<br>Chapter 2.1 --- Chemicals --- p.p.30<br>Chapter 2.1.1 --- Flavonoids --- p.p.30<br>Chapter 2.1.2 --- Plasmids --- p.p.30<br>Chapter 2.2 --- Mammalian cell culture --- p.p.31<br>Chapter 2.2.1 --- Maintenance of cells --- p.p.31<br>Chapter 2.2.2 --- Preparation of cell stock --- p.p.32<br>Chapter 2.2.3 --- Cell recovery from liquid nitrogen stock --- p.p.32<br>Chapter 2.3 --- Identification of estrogenic activity in flavonoids --- p.p.33<br>Chapter 2.3.1 --- Steady Glo Luciferase Assay --- p.p.33<br>Chapter 2.3.2 --- The Biorad Protein Assay kit (a modified Bradford method). --- p.p.33<br>Chapter 2.4 --- Viability Assay --- p.p.34<br>Chapter 2.5 --- ERE Luciferase reporter gene assay --- p.p.35<br>Chapter 2.5.1 --- Transient transfect ion of cell using lipofectamine PLUS reagent --- p.p.36<br>Chapter 2.5.2 --- Dual Luciferase Assay --- p.p.37<br>Chapter 2.6 --- ERα competitive binding ASSAY --- p.p.37<br>Chapter 2.7 --- Apoptotic death assay --- p.p.38<br>Chapter 2.8 --- Semi-quantitative RT-PCR Assay --- p.p.40<br>Chapter 2.8.1 --- "Isolation of RNA using TRIzol® Reagent (Life Technology,USA) " --- p.p.40<br>Chapter 2.8.2 --- Quantitation of RNA --- p.p.41<br>Chapter 2.8.3 --- First strand cDNA synthesis --- p.p.41<br>Chapter 2.8.4 --- PCR reactions --- p.p.43<br>Chapter 2.9 --- Flow Cytometry Analysis --- p.p.43<br>Chapter 2.10 --- Total triglyceride and cholesterol measurement --- p.p.44<br>Chapter 2.10.1 --- Determination of the total cholesterol --- p.p.45<br>Chapter 2.10.2 --- Determination of the total triglyceride --- p.p.46<br>Chapter 2.11 --- Manipulation of DNA and RNA --- p.p.46<br>Chapter 2.11.1 --- Transformation of DH5α --- p.p.46<br>Chapter 2.11.2 --- Mini preparation of plasmid DNA --- p.p.47<br>Chapter 2.11.3 --- Preparation of plasmid DNA using QIAGEN-tip 100 midi-prep kit --- p.p.48<br>Chapter 2.11.4 --- Preparation of plasmid DNA using QIAGEN-tip 10000 Giga-prep kit --- p.p.49<br>Chapter 2.11.5 --- Ethanol preparation of DNA and RNA --- p.p.50<br>Chapter 2.11.6 --- Agarose gel electrophoresis of DNA --- p.p.51<br>Chapter 2.12 --- Statistical methods --- p.p.52<br>Chapter CHAPTER 3 --- Estrogenic and antiproliferative activities on MCF-7 breast cancer cells by flavonoids<br>Chapter 3.1 --- Introduction --- p.p.53<br>Chapter 3.2 --- Results --- p.p.56<br>Screening of phytoestrogens for estrogenic activities on MELN cells --- p.p.56<br>Cell proliferation activity of phytoestrogens on MCF-7 and MDA-MA231 cells --- p.p.59<br>Estrogenic and antiestrogenic activity of phytoestrogens on ERα or erβ transfected hepg2 cells --- p.p.64<br>Chapter 3.3 --- Discussion --- p.p.73<br>Chapter Chapter 4 --- interaction of baicalein with estrogen receptors<br>Chapter 4.1 --- Introduction --- p.p.76<br>Chapter 4.2 --- Results --- p.p.78<br>Estrogen receptor competition assay --- p.p.78<br>ERE-Luciferase gene reporter assay --- p.p.82<br>Chapter 4.3 --- Discussion --- p.p.88<br>Chapter Chapter 5 --- baicalein and genistein display differential actions on er transactivation<br>Chapter 5.1 --- Introduction --- p.p.90<br>Chapter 5.2 --- Results --- p.p.92<br>Estrogenic and antiestrogenic activities of genistein and baicalein on ER transactivation --- p.p.92<br>Chapter 5.3 --- Discussion --- p.p.105<br>Chapter CHAPTER 6 --- APOPTOTIC EFFECTS OF BAICALEIN ON MCF-7 AND MDA-MB-231 CELL LINES<br>Chapter 6.1 --- Introduction --- p.p.107<br>Chapter 6.2 --- Results --- p.p.111<br>ER POSITIVE MCF-7 AND ER NEGATIVE MDA-MB-231 cell death assay --- p.p.111<br>"Bcl-2, Bax and PS2 mRNA expression " --- p.p.116<br>Arrest at sub G1 phase of MCF-7 by baicalein --- p.p.124<br>Chapter 6.3 --- Discussion --- p.p.127<br>Chapter CHAPTER 7 --- BAICALEIN CAN REDUCE INTRACELLULAR cholesterol and triglceride<br>Chapter 5.1 --- Introduction --- p.p.129<br>Chapter 5.2 --- Results --- p.p.130<br>Baicalein has beneficial effect on lipid metabolism --- p.p.130<br>Chapter 5.3 --- Discussion --- p.p.139<br>Chapter chapter 8 --- Summary --- p.p.140<br>BIBLIOGRAPHY --- p.p.142<br>APPENDIX 1 ABBREVIATIONS --- p.p.153<br>APPENDIX 2 PRIMER LISTS --- p.p.156<br>APPENDIX 3 REAGENTS AND BUFFERS --- p.p.157

Indiana University-Purdue University Indianapolis (IUPUI)<br>Aromatase inhibitors (AIs) are the current standard of care for the treatment of hormone receptor positive breast cancer in postmenopausal women. Nearly one-half of patients receiving AI therapy develop musculoskeletal toxicity that is characterized by joint and/or muscle pain and approximately one-fourth of patients discontinue their therapy as a result of musculoskeletal pain. Since there are no effective strategies for prevention or treatment, insight into the mechanisms of AI-induced pain is critical to improve treatment. However, there are few studies of AI effects in animal models of nociception. To determine whether AIs produce hypersensitivity in animal models of pain, I examined the effects of AI administration on mechanical, thermal, and chemical sensitivity in rats. The results demonstrate that (1) repeated injection of 5 mg/kg letrozole in male rats produces mechanical, but not thermal, hypersensitivity that extinguishes when drug dosing is stopped; (2) administering a single dose of 1 or 5 mg/kg letrozole in ovariectomized (OVX) rats also induces mechanical hypersensitivity, without altering thermal sensitivity and (3) a single dose of 5 mg/kg letrozole or daily dosing of letrozole or exemestane in male rats augments flinching behavior induced by intraplantar ATP injection. To determine whether the effects of AIs on nociceptive behaviors are mediated by activation or sensitization of peptidergic sensory neurons, I determined whether letrozole exposure alters release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons and from sensory nerve endings in rat spinal cord slices. No changes in basal, capsaicin-evoked or high extracellular potassium-evoked CGRP release were observed in sensory neuronal cultures acutely or chronically exposed to letrozole. Furthermore, letrozole exposure did not alter the ability of ATP to augment CGRP release from sensory neurons in culture. Finally, chronic letrozole treatment did not augment neuropeptide release from spinal cord slices. Taken together, these results do not support altered release of this neuropeptide into the spinal cord as mediator of letrozole-induced mechanical hypersensitivity and suggest the involvement of other mechanisms. Results from this dissertation provide a new experimental model for AI-induced hypersensitivity that could be beneficial in delineating mechanisms mediating pain during AI therapy.