Introdução: As variações da CYP2D6 estão associadas com o desfecho clínico das pacientes na terapia adjuvante do câncer de mama com tamoxifeno (TAM). Esta associação deve-se principalmente a hidroxilação do N-desmetiltamoxifeno (NDT) pela CYP2D6 a endoxifeno (EDF) que, por sua alta potência antiestrogênica, é o principal responsável pela eficácia terapêutica. O objetivo deste estudo foi avaliar a relação entre o genótipo e o fenótipo da CYP2D6 com os níveis de EDF e a razão metabólica [NDT]/[EDF] em uma população do Sul do Brasil. Métodos: Foram obtidas amostras de plasma em vale de 97 pacientes em terapia com o tamoxifeno. A genotipagem da CYP2D6 foi realizada com ensaio Luminex, com determinação de escores de atividade genotípica (EAG). As concentrações do TAM e seus metabólitos foram determinados por CLAE-DAD e classificadas em metabolizadores lentos (ML), intermediários (MI), rápidos com atividade diminuída (MR-D), rápidos com atividade rápida (MR-R) e ultra-rápidos (UR). A fenotipagem foi realizada através da determinação do dextrometorfano (DMT) e do dextrorfano (DTF) por CLAE-FL nas amostras de plasma coletadas três horas após administração oral de 33 mg de DMT. A atividade da CYP2D6 foi avaliada pela razão metabólica [DMT]/[DTF]. Resultados: A genotipagem da CYP2D6 mostrou prevalência de 4,1% de ML, 4,1% de MI, 49,5% de MR-D, 39,2% de MR-R e 3,1% de UR. O genótipo (EAG) foi significativamente correlacionado com o fenótipo ([DMT]/[DTF]), com uma associação moderada (rs= -0,463; p<0,001). As medianas das concentrações plasmáticas do TAM e seus metabólitos (ng mL-1) foram: TAM 57,17; HTF 1,01; EDF 6,21; NDT 125,50. Os níveis de EDF foram inferiores nos ML em comparação aos MR (p<0,05). O fenótipo apresentou maior associação, porém ainda moderada, com os níveis de EDF e [NDT]/[EDF] em comparação ao genótipo (r= -0,507 r=0,625, p<0,001 versus r= 0,356 r=0,516; p<0,01). Ao fenótipo da CYP2D6 foram atribuídas 26% da variabilidade dos níveis de EDF e 38 % das razões metabólicas [NDT]/[EDF], enquanto que ao genótipo foram atribuídas 12% e 27 %, respectivamente. Conclusão: A genotipagem e/ou fenotipagem da CYP2D6 não foram capazes de predizer completamente as concentrações de EDF. Desta forma, sugerimos que estudos futuros utilizem o monitoramento dos níveis de EDF durante a terapia com TAM, com o objetivo de avaliar a sua efetividade. / Background: An association between CYP2D6 variation and clinical outcomes among women with breast cancer treated with tamoxifen (TAM) has been demonstrated, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes. This association is mainly due the CYP2D6 mediated hydroxylation of N-desmethyltamoxifen (NDT) to yield endoxifen (EDF), which because of its high antiestrogenic potency, is the main responsible for the therapeutic efficacy of TAM. The aim of this study was to evaluate the relation of CYP2D6 genotyping and phenotyping with EDF levels and [NDT]/[EDF] metabolic ratio in breast cancer patients from South of Brazil under TAM therapy. Methods: Trough blood samples were collected from 97 patients. CYP2D6 genotyping was performed with a Luminex assay and calculation of Genotypic activity scores (GAS). Tamoxifen and metabolites EDF, NDT and 4-hidroxy-tamoxifen (HTF) were measured in plasma by HPLC-PDA. CYP2D6 phenotyping was performed by determination of dextromethorphan (DMT) and dextrorphan (DTF) by HPLC-FL at plasma collected three hours after oral administration of 33 mg of DMF. Phenotypes were given according to [DMT]/[DTF] metabolic ratio. Results: CYP2D6 genotyping indicated a prevalence of 4.1% PM, 4.1% IM, 49.5% EM-S, 39.2% EM-F and 3.1% UM. Genotype (GAS), was significantly correlated with phenotype ([DMT]/[DTF]), with a moderate association (rs= -0.463; p<0.001). Median plasma concentrations (ng mL-1) (N=97) were: TAM 57.17; HTF 1.01; EDF 6.21; NDT 125.50. EDF levels were lower in PM than in EM (p<0.05). Phenotype showed stronger, but still moderate, association with EDF and [NDT]/[EDF] than genotype (r= -0.507 r=0.625, p<0.001 versus r= 0,356 r=0.516, p<0.01). Phenotype accounted for 26% of the variability in EDF levels and 38 % of [NDT]/[EDF], while genotype for 12% and 27 %, respectively. Conclusion: CYP2D6 genotyping and/or phenotyping could not fully predict EDF concentrations. Monitoring EDF itself could be considered in further studies during TAM therapy in order to evaluate its efficacy

Le cancer du sein (CS) est le cancer le plus fréquent et la principale cause de décès par cancer chez les femmes. Par conséquent, il ya un besoin urgent de caractériser les acteurs moléculaires impliqués dans l'étiologie du cancer du sein, de la progression et de la résistance afin de développer de nouvelles solutions thérapeutiques pour améliorer le pronostique des patients. Le but de ce travail a été d'étudier un onco-métabolite produit à partir du cholestérol, identifié au laboratoire, et de caractériser son rôle et les enzymes régulant sa formation. Cet onco-métabolite est un promoteur de tumeur in vivo, qui stimule la croissance et l'invasion de différents sous-types de CS, y compris les plus agressifs. L'identification de cette nouvelle voie métabolique pourrait fournir de nouveaux marqueurs des CS et de l'efficacité des composés anti-cancéreux, ainsi que des thérapies innovantes permettant de bloquer la production de cet onco-métabolite. / Breast cancer (BC) is the most frequent cancer and the leading cause of cancer death in women. Therefore, there is an urgent need to characterize the molecular players involved in the etiology of breast cancers, progression and resistance in order to develop new therapeutic alternatives to improve the prognosis of patients. The present work focuses on the study of an onco-metabolite produced from cholesterol that has been identified in the laboratory and to characterize its role and the enzymes producing and regulating this onco-metabolite. This onco-metabolite is a tumor promoter that stimulates in vivo the progression and invasiveness of different subtypes of human BC, including the most aggressive ones. The identification of this new metabolic pathway will produce news markers for BC and of the efficacy of anti-cancer compounds, as well as innovative therapeutics to counter the production of this onco-metabolite.

Hot flashes are one of the most bothersome symptoms experienced by women who have undergone breast cancer treatment-induced menopause. This vasomotor symptom has been hypothesized to be responsible for decreased sleep quality. This study further investigated the relationship between hot flashes and sleep quality in this population. The convenience sample consisted of 30 women being seen at an outpatient clinic in a comprehensive cancer center in southwest Florida. All participants were between the ages of 36-65, had a diagnosis of breast cancer and were currently taking a selective estrogen receptor modulator for at least six weeks. The participants completed the Hot Flash Diary, Hot Flash Questionnaire, Hot Flash Related Daily Interference Scale, Pittsburgh Sleep Quality Index and a demographic form. The mean sleep score of the sample was 9.33 (SD= 4.4). Global sleep scores above five are indicative of poor sleep quality, and global sleep scores of eight or more have been linked to cancer-related fatigue. Sleep was strongly correlated with hot flash distress (r = .754, p. = .000) and hot flash severity (r = .718, p. = .000) and moderately correlated with hot flash interference (r = .507, p. = .004) and hot flash frequency while asleep (r = .680, p. = .000). The small sample size was a study limitation. However, study results do support findings from previous studies. This study addresses a symptom management problem that may give nurses better understanding of the experiences of their patients. These findings also may assist patients in helping their providers to understand the frustration they are experiencing with regard to their decreased sleep quality.

This body of research investigates the effects of sex and estrogen on higher brain functions, in general, and on working memory, in particular. A female advantage for object-location and verbal working memory has been reported, and estrogen supplementation facilitates performance of the same. Less is known about whether the female advantage is due to the verbalizability of the stimuli, and whether estrogen-suppression adversely affects performance on working memory and other neuropsychological domains sensitive to estrogen. Study 1 examined sex-related differences in young adults on an object-location working memory measure that varied in verbalizability of stimuli and task presentation (i.e., manual or computer); as expected, females performed better than males regardless of the verbalizability of the stimuli or task presentation. Study 2 examined sex-related differences in young adults on the n-back working memory task across verbal, spatial, and object conditions. Contrary to the hypotheses, there was no sex effect for the verbal version of the n-back task, and males actually performed better than females on the object version; as expected, males performed better than females on the spatial version. Study 3 investigated the effect of estrogen suppression in middle-aged and older adult females undergoing treatment for estrogenic breast cancer using the experimental working memory measures from Studies 1 and 2, and a comprehensive neuropsychological battery that included measures considered to be either sensitive (e.g., letter fluency) or insensitive (e.g., spatial ability) to the female advantage and the effects of estrogen. The estrogen suppression group performed more poorly than healthy age-matched controls on certain estrogen-sensitive measures (i.e., speeded visuomotor attention, speeded manual dexterity, and letter fluency), but unexpectedly, the groups did not differ on any of the memory measures presumed to be estrogen-sensitive. This body of research suggests that although certain working memory measures are sensitive to sex effects, the direction depends on the domain, and that estrogen suppression does not impact working memory in postmenopausal women but does adversely impact speeded performance measures.

Rhabdomyosarcoma (RMS) is a malignant soft-tissue sarcoma in children, accounting for about 40% of pediatric soft-tissue tumours. Five-year survival for metastatic RMS is only about 25%. Furthermore, there has been no significant improvement in RMS survival since 1975, pointing to a need for improved therapy. Previous work in our lab has shown that 4-hydroxytamoxifen (4OHT) leads to increased apoptosis and decreased viability in RMS cells. Expanding on this work, the current project aims to elucidate the mechanisms behind 4OHT-induced apoptosis in RMS cells, focusing on the roles of estrogen receptors (ER) and MAP kinases (MAPK). We found that: 1) 4OHT-induced apoptotic signaling was associated with increased MAPK phosphorylation, 2) Inhibition of MAPK protected cells against 4OHT, 3) Inhibition of ER also protected against 4OHT, and 4) ER inhibition blocked 4OHT-associated MAPK phosphorylation.

Rhabdomyosarcoma (RMS) is a malignant soft-tissue sarcoma in children, accounting for about 40% of pediatric soft-tissue tumours. Five-year survival for metastatic RMS is only about 25%. Furthermore, there has been no significant improvement in RMS survival since 1975, pointing to a need for improved therapy. Previous work in our lab has shown that 4-hydroxytamoxifen (4OHT) leads to increased apoptosis and decreased viability in RMS cells. Expanding on this work, the current project aims to elucidate the mechanisms behind 4OHT-induced apoptosis in RMS cells, focusing on the roles of estrogen receptors (ER) and MAP kinases (MAPK). We found that: 1) 4OHT-induced apoptotic signaling was associated with increased MAPK phosphorylation, 2) Inhibition of MAPK protected cells against 4OHT, 3) Inhibition of ER also protected against 4OHT, and 4) ER inhibition blocked 4OHT-associated MAPK phosphorylation.

Evidence from an abundant number of studies suggests that human female reproductive functions have become impaired over the past half century and that there might be a relationship between endocrine disrupting chemicals (EDCs) and reduced fertility. It is, however, not known by what mechanisms EDCs affect different reproductive functions such as endometrial receptivity, embryo implantation and placentation. The endometrium is continuously changing its morphological and functional properties, responding to cyclic changes of oestrogen and progesterone levels during the menstrual cycle. These changes include monthly preparation for embryo implantation through changed endometrial angiogenic activity and consequent changes in endometrial vasculature. Use of primary human endometrial endothelial cells (HEECs) in this work was evaluated as a possible screening tool for effects caused by EDCs on human endometrial vasculature and subsequently on various endometrial functions. In this study HEEC and endometrial stromal cells were isolated. HEECs were grown in monocultures, and together with stromal cells in co-cultures, and exposed to endocrine active substances. These were cadmium, which has oestrogenic properties, tamoxifen, with anti-oestrogenic effects, mifepristone, which is an anti-progestin, and bisphenol A, with oestrogenic properties. The effects were evaluated by using proliferation and viability assays, migration and tube formation assays, quantitative PCR (qPCR), immunohistochemistry and western blot. Cadmium affected the expression of angiogenesis-related genes, and caused different effects in HEECs cultured alone vs. HEECs co-cultured with stromal cells. Tamoxifen altered the expression of angiogenesis-related genes and reduced HEEC migration, thus having an anti-angiogenic effect. Mifepristone caused reduced formation of tubular structures in tube-formation assays involving HEECs co-cultured with stromal cells. Bisphenol A promoted tube formation in co-cultured HEECs which was related to changes in the expression of several angiogenesis-related genes as well as up-regulated expression of VEGF-D protein. In conclusion, we showed that EDCs have the ability to induce changes in endometrial angiogenic activity in vitro and may thus disturb normal endometrial functions related to fertility and pregnancy. HEECs grown in vitro may provide valuable information on the effects of EDCs on human endometrial functions. However, this model is not suitable as a large-scale screening tool.

This body of research investigates the effects of sex and estrogen on higher brain functions, in general, and on working memory, in particular. A female advantage for object-location and verbal working memory has been reported, and estrogen supplementation facilitates performance of the same. Less is known about whether the female advantage is due to the verbalizability of the stimuli, and whether estrogen-suppression adversely affects performance on working memory and other neuropsychological domains sensitive to estrogen. Study 1 examined sex-related differences in young adults on an object-location working memory measure that varied in verbalizability of stimuli and task presentation (i.e., manual or computer); as expected, females performed better than males regardless of the verbalizability of the stimuli or task presentation. Study 2 examined sex-related differences in young adults on the n-back working memory task across verbal, spatial, and object conditions. Contrary to the hypotheses, there was no sex effect for the verbal version of the n-back task, and males actually performed better than females on the object version; as expected, males performed better than females on the spatial version. Study 3 investigated the effect of estrogen suppression in middle-aged and older adult females undergoing treatment for estrogenic breast cancer using the experimental working memory measures from Studies 1 and 2, and a comprehensive neuropsychological battery that included measures considered to be either sensitive (e.g., letter fluency) or insensitive (e.g., spatial ability) to the female advantage and the effects of estrogen. The estrogen suppression group performed more poorly than healthy age-matched controls on certain estrogen-sensitive measures (i.e., speeded visuomotor attention, speeded manual dexterity, and letter fluency), but unexpectedly, the groups did not differ on any of the memory measures presumed to be estrogen-sensitive. This body of research suggests that although certain working memory measures are sensitive to sex effects, the direction depends on the domain, and that estrogen suppression does not impact working memory in postmenopausal women but does adversely impact speeded performance measures.

Conventional methods of chemotherapy requires novel therapy systems due to serious side effects and inefficiency of drug administration. In recent years many studies are carried out to improve drug delivery systems. Polymers are one of the most important elements for drug delivery research due to their versatility. By the discovery of dendritic polymers, drug delivery studies gained a new vision. Highly branched monodisperse structure, multiple sites of attachment, well-defined size and controllable physical and chemical properties make them efficient drug delivery systems. In this research hyperbranched dendritic polymers were sythesized and characterized for hydrophobic drug delivery. Dipentaerythritol which was used as core molecule, esterified with dimethylol propionic acid. Ricinoleic acid was esterified with the end groups of dimethylol propionic acid and hyperbranched resin (HBR) was formed. By considering the properties of HBR, hydrophobic tamoxifen and idarubicin were used for drug delivery study. The most efficient loading was determined as 73% for tamoxifen and 74% for idarubicin. Drug-HBR interactions and changes in properties of HBR were determined by FTIR, zeta potential and particle size measurements. FTIR results indicated that idarubicin chemically interacted with HBR while tamoxifen physically loaded to HBR. Drug delivery profile of HBR was studied in the absence and presence of lipase from Pseudomonas sp. and sodium dodecyl sulfate (SDS). Results revelaed that lipase and SDS increased the release rate of tamoxifen while idarubicin release rate was not affected. The effect of lipase was also tested for the degradation of HBR and it was indicated that lipase sustain a faster degradation. Finally toxicity of HBR and drug loaded HBR on MCF-7 breast cancer cell line was determined with XTT proliferation assay. Empty HBR did not cause significant toxicity on MCF-7 cells while drug loaded HBR was more toxic than free drug. By this study the efficiency of novel synthesized hyperbranched polymer in drug delivery was shown.

Tamoxifen is the most widely used endocrine therapy for both early and advanced estrogen receptor (ER) positive breast cancer patients. About half of the patients that initially respond to the antiestrogen become estrogen-independent and ultimately develop resistance to the treatment. The precise molecular mechanisms of tamoxifen resistance remain poorly understood. Dysregulation of microRNAs (miRNAs) has been frequently reported in breast cancer and linked to cancer development, progression and therapeutic response. To gain a more comprehensive picture of the miRNA regulatory network for modulating tamoxifen responsiveness, we examined global expression profiles of more than 600 miRNAs in a matched pair of tamoxifen-sensitive ZR75 and tamoxifen-resistant AK47 breast cancer cell lines using TaqMan Low Density Array (Applied Biosystems). Under 4-hydroxytamoxifen treatment, 102 miRNAs displayed differential responses between the sensitive cells and the resistant cells. At basal levels, upregulation of 32 miRNAs and downregulation of 75 miRNAs were observed in the resistant cells as compared to the sensitive cells. Among the 9 miRNAs of significant differential expression selected for validation, expression profiles of the 7 miRNAs could be reproduced. Of these, 4-hydroxytamoxifen treatment greatly increased miR-449a/b expression in sensitive ZR75 cells, whereas miR-449a/b expression was significantly reduced in resistant AK47 cells at basal levels, which was further confirmed in a panel of tamoxifen-resistant breast cancer cell lines. Such downregulation of miR-449a/b in the resistant cells was partially attributed to DNA methylation-mediated repression of miR-449a/b. Notably, miR-449a/b expression exhibited a significant positive correlation with ER-α status (miR-449a: P=0.006, miR-449b: P=0.013) and progesterone receptor (PR) status (miR-449a: P=0.010, miR-449b: P=0.021), and a prominent inverse association with tumor grade in 61 breast cancer tissues (miR-449a: P=0.001; miR-449b: P=0.009). Also, breast cancer patients with high miR-449a/b expression tended to have increased disease-free survival (miR-449a: P=0.019; miR-449b: P=0.117). To further support the tumor suppressor function of miR-449, stable miR-449b overexpression in the resistant cells reduced cell proliferation. More intriguingly, restoring miR-449b expression increased sensitivity to 4-hydroxytamoxifen-induced apoptosis via suppression of AKT activity without restoring ER-α expression. In contrast, miR-449a/b knockdown reduced ER-α and PR expression, but enhanced phosphorylation of AKT, extracellular signal-regulated kinase- 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and also ER-α at serine 167 and serine 118 residues. Furthermore, we demonstrated c-Myc is a target gene of miR-449 as confirmed by bioinformatics and experimental analyses. Computational algorithms predicted a highly conserved miR-449a/b binding site within C-MYC 3’untranslated region (3’UTR). Compared to the parental sensitive cells, c-Myc was overexpressed in the resistant cells. Forced expression of miR-449b suppressed c-Myc protein level. To further support the notion that c-Myc is a direct target of miR-449, interactions between miR-449b and C-MYC 3’UTR were confirmed by co-expression of miR-449b and c-Myc expression constructs and luciferase reporter assay. Taken together, our data strongly suggest the critical role of miR-449 in modulating altering response to tamoxifen via targeting c-Myc. Suppression of miR-449 repressed genomic ER action and concomitantly activated non-genomic ER pathways. These findings may provide insights to improve breast cancer management and open a wide avenue for therapeutic interventions for overcoming tamoxifen resistance. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy