Dissertations / Theses on the topic 'MCSF receptor'

The testes are glands with endocrine and exocrine functions, the latter being characterized by sperm formation, or spermatogenesis. This is under the control of a complex system involving endocrine and paracrine signalization. In view of an interrelationship between the expression of c-Fos and estrogen receptor beta (ERbeta) proteins, this investigation evaluated the expression of the protooncogene c-fos and the immunolocalization of c-Fos, phosphorylated c-Fos and ERbeta proteins in the human testis. Testis tissue was obtained from 12 men undergoing orchiectomy as a treatment for prostate cancer. These patients had received no hormonal, chemo or radiation therapy before the operations. Tissues were stained by immunohistochemistry using the avidin-biotin-peroxidase method, and c-fos RNAm expression was assessed with reverse transcriptase- polymerase chain reaction (RT-PCR). The protooncogene c-fos was expressed in the human testis and both forms of c-Fos proteins were immunoreactive, mainly in germ cells (spermatogonia, spermatocytes and spermatids) and Sertoli cells. ERbeta was primarily present in somatic cells (Leydig, Sertoli and myofibrillar cells). Based on these results, we hypothesized two mechanisms for estrogen actions over spermatogenesis. The first would be mediated by Sertoli cells, where estrogens could alter either gene expression or the transcriptional activity of c-Fos protein. The other is an indirect mechanism, representing the interrelationship between somatic and germ cells. Somatic cells, under estrogen influence, could modify germ cell functions by paracrine/juxtacrine factors, which would change gene expression or the transcriptional activity of c-Fos protein.
Os testículos são glândulas com função endócrina e exócrina, sendo que esta última se caracteriza pela formação de gametas masculinos, ou seja, a espermatogênese. Esta se encontra sob a influência de um complexo sistema de sinalização endócrina (sistêmica e parácrina). Considerando uma possível inter-relação entre as proteínas c-Fos e receptor de estrogênios beta (ER-beta), este trabalho verificou a expressão do proto-oncogene c-fos e a imunolocalização das proteínas c-Fos, c-Fos fosforilada e ER-beta no parênquima testicular humano. A amostra foi constituída por 12 homens férteis, portadores de adenocarcinoma prostático, com indicação terapêutica de bloqueio hormonal cirúrgico pela orquiectomia. Nenhum destes havia sido previamente submetido a tratamento hormonal, quimioterapia ou radioterapia. O material foi processado para o estudo imunohistoquímico com o método da avidina-biotina-peroxidase e avaliação da expressão gênica por transcrição reversa e reação em cadeia da polimerase (RT-PCR). A expressão do proto-oncogene c-fos foi comprovada no testículo humano, e as proteínas c-Fos e c-Fos fosforilada foram localizadas principalmente no epitélio seminífero, tanto nas células germinativas (espermatogônias, espermatócitos e espermátides) quanto nas células de Sertoli. O ER-beta foi expresso principalmente em células somáticas (células de Leydig, Sertoli e mióides). A distribuição encontrada das proteínas c-Fos e ER-beta nos permite supor uma inter-relação entre estas; seja pela ação estrogênica na célula de Sertoli, induzindo a expressão local de c-fos, seja indiretamente, pelos efeitos que o estímulo estrogênico sobre as células somáticas poderia ter sobre a expressão de c-fos no epitélio germinativo.

Several substances may modulate reflex cardiovascular activity, such reninangiotensin system peptides. The tecidual renin-angiotensin system is involved in regulation of cardiovascular structures and functions and, consequently in long-term blood pressure control, contributing in a significant way to maintenance and development of high peripheral resistance and vascular hypereactivity found in the essential and experimental hypertension. Studies about the renin-angiotensin influences on neural control of BP show that Ang II reduces baroreflex sensitivity, reduces the Bezold-Jarisch reflex sensitivity and increases the chemoreflex responses. Because known that several Ang-(1-7) actions are opposite to Ang II actions, mainly about cardiac and vascular control, as well baroreflex sensibility, it is reasonable to suppose that the heptapeptide Ang-(1-7) acting on Ang-(1-7)s Mas receptor, also to take part of the Bezold-Jarisch reflex and the chemoreflex. To have the opportunity to use genetic change animals as for Mas receptor expression, we asses the hypothesis that Ang-(1-7), via Mas receptor, to contribute for the reflex cardiovascular modulation, in mice with Mas receptor deletion ou Mas receptor overexpression at brain. In sequence, considering the evidences of counter-regulatory actions between Ang II/Ang-(1-7), and the possibility to interaction between Mas/AT1 receptors, we also to asses the hypothesis that Mas receptor deletion to change the Ang II effects, because the absence to counter-regulatory effects to Ang-(1-7), via Mas receptor. We use control (Wild-Type) and Mas receptor expression changes mice, Knockout Mas receptor mice (KO-Mas) and overexpression in brain Mas receptor (NSE-Mas), with background C57BL6J and FVBN. We showed in this study, through genetic manipulation of the Mas receptor expression, the effective participation of Angiotensin-(1-7) in the xiii cardiovascular control. Mas knockout mice (FVBN background) awake show high basal MAP values in comparison with control mice (118 ± 1 vs. 109 ± 2 mmHg, p<0.01), but not show significant changes in basal HR values (615 ± 30 vs. 648 ± 13 bpm). The deletion of Mas receptor decreases baroreflex bradycardia (0.78 ± 0.44 vs. 1.3 ± 0.14 ms/mmHg, p<0.05), increases baroreflex tachycardia (1.63 ± 0.4 vs. 0.80 ± 0.13ms/mmHg, p<0.05), decreases the bradycardic and hypotensive response of Bezold- Jarisch reflex (-17 ± 5 vs. -45 ± 6 mmHg e -212 ± 36 vs. -391 ± 29 bpm, FB 0.5Ng/5Nl, p<0.01) and increases the reflex responses observed by chemoreflex stimulation (20 ± 3vs. 12 ± 0.8 mmHg e -250 ± 74 vs. -52 ± 26 bpm, KCN 2.5Ng/5Nl, p<0.05) in awake FVBN background mice. In accordance with changes describe, we showed that Mas knockout receptor mice had reduce tachycardic effect produce by methilatropine administration (23 ±4 vs. 70 ± 19 bpm, p<0.05) and increase bradycardic effect induced by atenolol administration (-181 ± 10 vs. -115 ± 23 bpm, p<0.05). When we measure transgenic mice with overexpression of Mas receptor in brain (NSE-Mas), demonstrate that Ang-(1-7), acting in CNS, participate of basal and reflex cardiovascular variables control. The Mas receptor overexpression in brain mice induces increases of basal MAP (116 ± 3 vs. 109 ± 2 mmHg, p<0.05) and not change basal HR values (641 ± 18 vs. 648 ± 13 bpm). About bradycardic barorefelx response, we showed that Mas receptor overexpression in brain promoves increase this response (2.03 ± 0.33 vs. 1.3 ± 0.14 ms/mmHg, p<0.05). After A- 779, Ang-(1-7) antagonist, ICV administration, despite did not show significant changes in MAP and HR basal values in NSE-Mas and control mice, we show that bradycardic (1.17 ± 0.14 vs. 1.92 ± 0.37 ms/mmHg, p<0.05), and tachycardic (0.49 ± 0.11 vs. 0.94 ± 0.32 ms/mmHg, p<0.01), baroreflex responses were significantly decrease only in control FVBN background awake mice. In this study, we highlight the important interaction Ang-(1-7)/Mas couter-balancing the classic actions of Ang II/AT1 in the cardiac autonomic activity maintenance, as reflexes and basal cardiovascular responses control. These results join with literature reports, contribute to physiologic relevancy of Ang-(1-7), acting on the Mas receptor, in the cardiovascular function control.
Várias substâncias podem modular a atividade cardiovascular reflexa, entre elas os peptídeos do SRA. O SRA tecidual está envolvido na regulação da estrutura e função cardiovascular e, consequentemente no controle a longo prazo da PA, contribuindo de forma significativa para o desenvolvimento e manutenção da alta resistência periférica e hiper-reatividade vascular encontradas em várias formas de hipertensão essencial e experimental. Os estudos sobre a influência do SRA sobre o controle neural da PA demonstram que a Ang II diminui a sensibilidade do barorreflexo, diminui a sensibilidade do reflexo de Bezold-Jarisch e facilita a resposta quimiorreflexa. Sabendo-se que várias ações da Ang-(1-7) são opostas àquelas observadas pela Ang II, principalmente no que se refere aos seus efeitos sobre o controle cardíaco e vascular, bem como sobre a sensibilidade barorreflexa, é razoável supor que o heptapeptídeo Ang-(1-7), agindo em seu receptor Mas, também participe da regulação do reflexo Bezold-Jarisch e do quimiorreflexo. Com a possibilidade de utilizar animais geneticamente modificados quanto à expressão do receptor Mas, avaliamos a hipótese de que a Ang-(1-7), via receptor Mas, contribui para a modulação das respostas cardiovasculares reflexas, em camundongos com deleção do receptor Mas ou com superexpressão deste receptor no cérebro. Na seqüência, considerando as evidências de ações contra-regulatórias entre Ang II/Ang-(1-7), e a possibilidade de interação dos receptores Mas/AT1, também avaliamos em nosso estudo a hipótese de a deleção do receptor Mas alterar os efeitos mediados pela Ang II, em decorrência da ausência dos efeitos contra-regulatórios mediados pela Ang-(1-7), via receptor Mas. Utilizamos camundongos controle (Wild Type) e com alterações da expressão do receptor da Ang-(1-7), receptor Mas: com deleção do receptor da Ang-(1-7) x Mas (KO-Mas) e com superexpressão deste receptor no cérebro (NSE-Mas), de duas linhagens diferentes (C57BL6J e FVBN). Demonstramos neste estudo, através da utilização de camundongos submetidos à manipulação da expressão gênica do receptor Mas, a efetiva participação da Ang-(1-7) via receptor Mas na manutenção e controle das variáveis cardiovasculares. Camundongos com deleção do receptor Mas da linhagem FVBN não anestesiados apresentam valores de PAM basais elevados em relação aos animais controle (118 ± 1 vs. 109 ± 2 mmHg, p<0.01), mas não apresentam alterações significativas nos valores basais de FC (615 ± 30 vs. 648 ± 13 bpm). A deleção do receptor Mas atenua a bradicardia barorreflexa (0.78 ± 0.44 vs. 1.3 ± 0.14 ms/mmHg, p<0.05), facilita a taquicardia barorreflexa (1.63 ± 0.4 vs. 0.80 ± 0.13 ms/mmHg, p<0.05), atenua as respostas hipotensora e bradicárdica que constituem o reflexo de Bezold-Jarisch (-17 ± 5 vs. -45 ± 6 W PAM, mmHg, e -212 ± 36 vs. -391 ± 29 W FC, bpm, FB 0.5Ng/5Nl, p<0.01) e facilita as respostas reflexas observadas após estimulação do quimiorreflexo (20 ± 3 vs. 12 ± 0.8 W PAM, mmHg, e -250 ± 74 vs. -52 ± 26 W FC, bpm, KCN .5Ng/5Nl, p<0.05) em camundongos da linhagem FVBN, não anestesiados. Em acordo com as alterações descritas, observamos que a deleção do receptor Mas reduz o efeito taquicárdico promovido pela administração de metilatropina (23 ± 4 vs. 70 ± 19 W FC, bpm, p<0.05) e aumenta o efeito bradicárdico promovido pela administração de atenolol (-181 ± 10 vs. - 115 ± 23 W FC, bpm, p<0.05). Utilizando camundongos transgênicos que superexpressam o receptor Mas no cérebro (NSE-Mas), observamos que a Ang-(1-7), agindo no SNC, participa do controle das variáveis cardiovasculares basais e reflexas. A superexpressão do receptor Mas no cérebro induz elevação dos valores basais de PAM (116 ± 3 vs. 109 ± 2 mmHg, p<0.05) e não altera os valores basais de FC (641 ± 18 vs. 648 ± 13 bpm). Com xi relação à resposta barorreflexa, observamos que a superexpressão do receptor Mas no cérebro promove facilitação da bradicardia barorreflexa (2.03 ± 0.33 vs. 1.3 ± 0.14 ms/mmHg, p<0.05). Para avaliar a contribuição da Ang-(1-7) endógena, administramos ICV o antagonista da Ang-(1-7), A-779, e apesar de não observarmos alterações significativas nos valores de PAM e FC basais dos camundongos NSE-Mas e controle, observamos que as respostas bradicárdica (1.17 ± 0.14 vs. 1.92 ± 0.37 ms/mmHg, p<0.05), e taquicárdica (0.49 ± 0.11 vs. 0.94 ± 0.32 ms/mmHg, p<0.01), barorreflexas foram significativamente reduzidas nos camundongos controle. Considerando os resultados do presente estudo, destacamos a importância da interação Ang-(1-7)/Mas contrabalanceando as ações clássicas da Ang II/AT1 para a manutenção da atividade autonômica cardíaca, assim como na modulação das respostas cardiovasculares reflexas e na manutenção dos valores basais de PA. Estes resultados associados aos dados da literatura, corroboram a relevância fisiológica da Ang-(1-7), agindo via receptor Mas, no controle da função cardiovascular.

The metabolic syndrome, also known as insulin resistance syndrome, is characterized by the variable coexistence of obesity, insulin resistance, dislipidemy and hypertension. The angiotensin-(1-7) presents an important contaregulatory role inside Renin Angiotensin System, opposing some times to angiotensina II effects. It has been shown that G protein-coupled receptor, Mas, mediates many actions of angiotensin-(1-7). We have recently observed that Mas-knockout male mice (Mas-/-) in the pure, FVB/N genetic background, presents elevated blood pressure levels and endothelial dysfunction, alterations present in the metabolic syndrome. The aim of this study was to ascertain whether genetic deletion of Mas also changes lipidic and glycemic profile and the mechanisms of these alterations. Ten weeks old Mas-/- and WT mice were used. Curves of plasma glycemia versus time were built after intraperitoneal application of insulin (0.75U/Kg BW) or glucose (2g/Kg BW). After sacrifice, the tissues were weighted and reserved for western blotting and Real-Time PCR. The lipidic profile and the plasma levels of leptin and adiponectin were analyzed using ELISA kits and TGF-â, angiotensinogen and TNF-á mRNA expression was analyzed by Real Time PCR. Despite of having normal body weight (24.7 ± 0.35 vs 24.8± 0.24 g in WT), young Mas-/- mice presented a marked increase in the fat tissue mass (epididimal= 1.704 ± 0.1516 vs 1.150 ± 0.1259 % of BW in WT and retroperitoneal= 0.6747 ± 0.08576 vs 0.3781 ± 0.04575 % of BW in WT). In addition, these animals presented a state of insulin resistance and glucose intolerance as well as an increase in the fasting glycemia levels (86.6 ± 6.43 vs 56.40 ± 4.98 mg/dl in WT). Furthermore, a significant increase in total cholesterol (92.2 ± 3.65 vs 74.6± 5.67 mg/dl in WT) and triglycerides (70.6 ± 13.3 vs 41.4± 4.07 mg/dl in WT) levels were observed. Part of these alterations can be explained by the increase in the leptin plasma levels (1.3 ± 0.25 vs 0.73 ± 0.17 ng/ml in WT) and the decreased Glut4 receptor protein in Mas-/- adipose tissue. The mRNA expression of TGF-â and angiotensinogen was increased in Mas-/- adipose tissue, while the expression of TNF-á, the food intake and adiponectin plasma levels, were not altered. These results show that Mas deficiency in FVB/N mice leads to dramatic changes in glicemic and lipidic metabolism, inducing a metabolic syndrome- like state.
A síndrome metabólica, também conhecida como síndrome de resistência à insulina, é caracterizada pela coexistência variável de obesidade, hiperinsulinemia, dislipidemia e hipertensão. A angiotensina-(1-7) apresenta um importante papel contraregulatório dentro do Sistema Renina Angiotensina, se opondo, na maioria das vezes, aos efeitos da angiotensina II. Tem sido demonstrado que o receptor acoplado a proteína G, Mas, medeia várias ações da angiotensina-(1-7). Observamos recentemente que camundongos machos knockout para o receptor Mas (Mas-/-) com background genético FVB/N, apresenta pressão sanguínea elevada e disfunção endotelial, alterações presentes no quadro de síndrome metabólica. O objetivo desse estudo foi verificar se a deleção genética do receptor Mas altera o perfil lipídico e glicêmico desses animais e os mecanismos envolvidos nesse processo. Camundongos WT e knockout machos com aproximadamente dez semanas de vida foram utilizados. Curvas de glicemia pelo tempo foram construídas após aplicação intraperitoneal de insulina (0.75U/Kg) ou glicose (2g/Kg). Após o sacrifício os tecidos foram pesados e reservados para western blotting e Real-Time PCR. O perfil lipídico e os níveis plasmáticos de leptina e adiponectina foram avaliados utilizando kits de ELISA e a expressão do mRNA do TGF-â, angiotensinogênio e do TNF-á foram analisados pela técnica de Real-Time PCR. Apesar de apresentar peso corporal igual ao do controle (24.7 ± 0.35 vs 24.8± 0.24 g no WT), os camundongos Mas-/- jovens apresentaram marcante aumento no peso do tecido adiposo (epididimal= 1.704 ± 0.1516 vs 1.150 ± 0.1259 % do PC no WT e retroperitoneal= 0.6747 ± 0.08576 vs 0.3781 ± 0.04575 % do PC no WT). Além disso, esses animais apresentam resistência a insulina e maior intolerância a glicose, bem como um aumento na glicemia de jejum (86.6 ± 6.43 vs 56.40 ± 4.98 mg/dl no WT). Também foram encontrados aumentos significativos nos níveis plasmáticos de colesterol total (92.2 ± 3.65 vs 74.6± 5.67 mg/dl no WT) e triglicérides (70.6 ± 13.3 vs 41.4± 4.07 mg/dl no WT). Parte dessas alterações podem ser explicadas pelo aumento nos níveis séricos de leptina (1.3 ± 0.25 vs 0.73 ± 0.17 ng/ml no WT) e pela diminuição na expressão protéica do receptor Glut-4 no tecido adiposo epididimal dos Mas-/-. A expressão do RNA mensageiro do TGF-â e do angiotensinogênio estão aumentados no tecido adiposo, enquanto a expressão do TNF-á, o consumo de comida e os níveis plasmáticos de adiponectina, não estão alterados. Juntos, esses dados indicam um importante papel do receptor Mas na função cardiovascular e metabólica em camundongos FVB/N e sugerem um quadro de síndrome metabólica nos camundongos knockout Mas.

Angiogenesis depends on a complex network of cells and mediators. The activation or blockade of cannabinoid receptors showed to be an interesting pharmacological strategy to attenuate the angiogenic and/or inflammatory response in many experimental models. Here we investigated how this strategy may interfere in inflammatory angiogenesis. Polyester-polyurethane sponges were implanted in C57BL/6 mice. Animals received daily doses (10 mg/kg, s.c.) of the cannabinoid antagonists SR141716A (CB1) and SR144528 (CB2), separately, or 3 and 10 mg/Kg (30 or 100 ìg/mice, for the local treatment) (s.c.) of the cannabinoid CB1/CB2 agonist WIN 55212-2, for 7 or 14 days. The implants were then collected for ELISA, hemoglobin, myeloperoxidase and N-acetylglucosaminidase measurements, used as indexes for angiogenesis, neutrophil and macrophage accumulation, respectively. Histological analysis was also conducted. CB1 or CB2 receptor antagonist treatment was able to reduce cellular influx to the sponge at 7 and 14 days, with distinctive pattern for macrophages and neutrophils. The CB1/CB2 agonist also reduced cellular influx. Both treatments affected angiogenesis. These alterations were accompanied by changes in the levels of TNF-á, VEGF, CXCL1-3/KC, CCL2/JE and RANTES, depending on the treatment. All changes correspond to a similar pattern in the histological analysis. The blockade or activation of cannabinoid receptors showed to be effective in reducing inflammatory and angiogenic responses. Altough acting in a similar way, levels of cytokines, chemokines and endocannabinoids may help explain this paradoxal response. Partial agonism / inverse agonism activity and receptor desensitization is another explanation for these responses. Keywords: angiogenesis, inflammation, cannabinoid receptors, cytokines
A angiogênese é controlada por uma complexa rede de células e mediadores. A ativação ou bloqueio de receptores canabinóides demonstram ser uma interessante estratégia farmacológica para atenuar a resposta angiogênica e/ou inflamatória em vários modelos experimentais. Aqui investigamos como esta estratégia pode interferir na angiogênese inflamatória. Esponjas de polyester-poliuretana foram implantadas em camundongos C57BL/6. Os animais receberam doses diárias (10 mg/Kg, s.c.) dos antagonistas canabinóides SR141716A (CB1) and SR144528 (CB2), separadamente, ou 3 e 10 mg/Kg (30 ou 100 g/animal, para o tratamento local) (s.c.) do agonista CB1/CB2 WIN 55,212-2, por 7 ou 14 dias. Os implantes foram coletados para análises por ELISA, hemoglobina, mieloperoxidase e N-acetilglicosaminidase, utilizadas, respectivamente, como index para mediadores protéicos, angiogênese e acúmulo de neutrófilos e macrófagos, respectivamente. O tratamento com os antagonistas CB1 ou CB2 levou à redução do influxo celular para a matriz esponjosa nos dias 7 e 14, com padrões distintos para macrófagos e neutrófilos. O agonista CB1/CB2 também reduziu o influxo celular. Ambos os tratamentos interferiram na angiogênese. Estas alterações foram acompanhadas por mudanças nos níveis de TNF-, VEGF, CXCL1-3/KC, CCL2/JE e RANTES, dependendo do tratamento. Todas as mudanças apresentam padrões similares na análise histológica. A ativação ou bloqueio de receptores canabinóides parece ser efetivo em reduzir as respostas angiogênica e inflamatória. Embora agindo de forma similar, níveis de citocinas, quimiocinas e endocanabinóides podem explicar esta resposta paradoxal. Desensibilização dos receptores e atividade agonista parcial / agonista inverso são outras explicações plausíveis para estas respostas.

Epilepsies are brain disorders characterized by recurrent and spontaneous crises. They are caused by hyper-synchronized discharges, localized or generalized through the brain. Their high incidence and prevalence are responsible for both psycho and social impact. An important class of epilepsy is the reflex epilepsy, initiated by exposure to sensory stimulus. The audiogenic crises, a modality of reflex epilepsy, are related to dysfunction of the auditory pathway. The selection of WAR (Wistar Audiogenic Rats) lineage is based on crises susceptibility induced due to the exposure to the high intensity sound stimulus. Nevertheless these animals show greater sensibility to crises when exposed to other convulsive agents such as electroshock, pentyleneterazole and pilocarpina, suggesting changes on their neuronal network.We study the excitatory post-synaptic potential (EPSP) pattern in the presence and absence of picrotoxin (which blocks the GABAA receptors) in the hippocampus and amygdala of WAR lineage. We also analyze the induction of plastic modifications in the lateral amygdala, in presence or absence of picrotoxin. The results show a reduced response to the toxin, suggesting alteration on the GABAA tonus. In the hippocampus, this points to a generalized dysfunction of the neuronal substrates, but there is no evidence of involvement of such structure in audiogenic crises. Our results also show a tendency to increase in the basal response when compared to the control, suggesting a neuronal circuit more excitable and prone to epileptic crises. We did not observe a low or even standing potentiation in the amygdala, probably caused by its increased and saturated basal response and by a reduced excitatory modulation. In presence of picrotoxin LTP was not observed for WAR or Wistar group. The inhibitory effect of picrotoxin in EPSP on the amygdala is followed by a recovery response to basal levels, similar to the increase observed for LTP. We conclude that WAR show a GABAA system alteration leading to a hyperexcitability of their neuronal circuits. These animals also behave as resistant Wistar rats when their GABA A system is blocked.
As epilepsias são desordens do cérebro manifestadas por crises epilépticas espontâneas e recorrentes. São causadas por descargas neuronais hipersincronizadas, focais ou generalizadas no cérebro. Apresentam altas prevalência e incidência, causam grande impacto psico-social e físico ao seu portador. Uma classe importante de epilepsia é a que engloba as crises reflexas, que são desencadeadas pela exposição do indivíduo suscetível a um estímulo sensorial, em muitos casos bastante específico. A crise audiogênica é uma forma de epilepsia reflexa que está relacionada a disfunção da via auditiva. No entanto, apesar de a seleção da linhagem dos WAR (ratos wistar audiogênicos) ter sido feita pela susceptibilidade dos animais em desencadearem crise quando expostos a um estímulo sonoro de alta intensidade, esses animais apresentam maior sensibilidade, também a desencadearem crises quando expostos a outros agentes convulsivantes como eletrochoque, petilenotetrazol e pilocarpina. Sugerindo a existência de mecanismos que tornam suas redes neurais, de uma forma geral, hiperexcitáveis e hipersincrônicas. Neste trabalho avaliou-se o padrão do Potencial Excitátorio Pós Sináptico (PEPS) na presença e ausência de picrotoxina, bloqueador do sistema GABA A (ã-acído aminobutirico), em dois substratos neurais de WAR: em hipocampo, local onde o GABA exerce efeito inibitório e em amigdala lateral, onde o GABA exerce potente modulação excitatória. Avaliou-se também a capacidade da geração de modificações plásticas na amígdala lateral de WAR, na presença e na ausência de picrotoxina. Observamos baixa responsividade à picrotoxina tanto em hipocampo quanto em amigdala de WAR; sugerindo haver alteração do tônus GABAA nestes dois substratos neurais. A presença desta alteração em hipocampo de WAR, sugere uma disfunção generalizada nos substratos destes animais, uma vez que não há evidências sobre o envolvimento desta estrutura nas crises audiogênicas. Nossos resultados também demonstram uma tendência no aumento das respostas basais nestas duas estruturas dos WAR quando comparadas às dos wistar controles. Sugerindo um circuito neural 2 hiperexcitável e mais propenso à geração de crises epilépticas. Não obtivemos potenciação estável e duradoura em amigdala de WAR, provavelmente devido à sua resposta basal já aumentada e saturada, e devido a uma baixa modulação excitatória neste local. Após adição de picrotoxina não observamos LTP em nenhum dos grupos de animais estudados. Observamos que a ação inibitória da picrotoxina nos PEPS, em amigdala, é seguida por uma recuperação da resposta a níveis basais, aumento semelhante ao ocorrido na potenciação de longo prazo. Os resultados nos permitem concluir que os WAR apresentam alterações no sistema GABAA que favorecem a hiperexitabilidade dos seus circuitos neuronais e que estes animais se comportam como ratos wistar resistentes quando estão com o sistema GABAA bloqueado.

The endogenous lipid, platelet activating factor (PAF), is usually considered a pro-angiogenic mediator due to its exogenous in vitro and in vivo effects and to studies with PAF receptor (PAFR) antagonists in vivo. In this study, mice with gene inactivation of the receptor for PAF (PAFR-KO) well used to analyze a range of activities exhibited by this mediator. Two experimental designs were used: the sponge implantation and the growth of solid tumors (Ehrlich and colon) to assess neovascularization, inflammation, cytokine production. Angiogenesis as determined by morphometric analysis and hemoglobin content was significantly higher in the implants and colon tumor of knock out (KO) mice compared with the wild type (WT). VEGF content in the tumors was also, higher in these animals. The levels of TNF-alpha were increased in Ehrlich tumor in KO mice relative to the WT group. Neutrophils and macrophages accumulation, as determined by myeloperoxidase (MPO) and Nacetylglucosaminidase (NAG) were decreased in all proliferating tissues in KO animals, supporting the pro-inflammatory effects of endogenous PAF. The levels of the pro-inflammatory chemokines did not follow the same pattern. The growth of colon tumor was not different between the KO and WT mice. However, Ehrlich tumor was six fold bigger in PAFR-KO mice than in WT animals. We have shown that inflammation and angiogenesis in PAFR-KO mice are not necessarily causal events and propose that PAF may be an important endogenous inhibitor of new blood vessels formation and Ehrlich tumor development.
O lipídio endógeno, fator ativador plaquetário (PAF), é comumente considerado um mediador pró-inflamatório e pró-angiogênico com base na aplicação exógena deste composto e antagonistas de seus receptores in vitro e in vivo. Neste trabalho, usando camundongos com inativação do gene (nocaute (KO)) para o receptor do PAF (PAFR-KO) foram avaliadas várias atividades deste mediador em dois modelos de angiogênese (modelo de implante de esponjas; angiogênese inflamatória e os tumores sólidos de Ehrlich e cólon; angiogênese tumoral). Nestes modelos foram caracterizadas a neovascularização, a inflamação e a produção de citocinas. Além disso, o crescimento dos tumores sólidos nestes animais foi determinado. A angiogênese, avaliada por análise morfométrica nos implantes e pela dosagem do conteúdo de hemoglobina, nos dois tecidos estava aumentada tanto nos implantes como no tumor de cólon dos animais nocaute. Níveis aumentados do VEGF foram predominantes nos tumores dos animais nocaute (compatível com o aumento da angiogênese) e os do TNF-alfa variaram entre os tecidos avaliados nos dois grupos de animais. O acúmulo de neutrófilos e macrófagos determinados pela atividade das enzimas mieloperoxidade (MPO) e N-acetilglucosaminidase (NAG) respectivamente, nos implantes e tecidos tumorais foram significativamente menores nos animais PAFR-KO, confirmando os efeitos pró-inflamatórios do PAF. Os níveis da quimiocina KC foram maiores no implante dos animais nocaute. Nos tumores, esta quimiocina foi menor apenas no tumor de Ehrlich nestes animais. Não houve diferença entre os pesos dos tumores de cólon nos dois grupos de animais. No entanto, o crescimento do adenocarcinoma de Ehrlich no período de 25 dias foi seis vezes maior nos animais KO comparados aos selvagens. Os resultados deste estudo mostram que a inflamação e a angiogênese em camundongos PAFR-KO não são eventos causais e propomos que o PAF endógeno pode ser um importante mediador inibitório da neo-formação vascular e do desenvolvimento do tumor sólido de Ehrlich.

Steroid receptor coactivators modulate the final outcome of hormone induced gene transcription by steroid receptors. E6-associated protein (E6-AP), an E3 ubiquitin ligase, acts as a coactivator of steroid receptors, including estrogen receptor (ER). In this study, we elucidated the contribution of E6-AP to ER-dependent gene transcription in breast cancer cells. siRNA-mediated knockdown of E6-AP abrogates transcription of classic ER target genes, GREB1 and pS2, suggesting that E6-AP is essential for normal transactivation function of ER. In order to understand the global influence of E6-AP in ER-dependent gene transcription, we used gene expression microarrays under E6-AP knockdown conditions to identify ER target genes which are regulated by E6-AP. Our microarray analysis revealed 455 genes which are differentially regulated by E6-AP. Pathway analysis revealed that E6-AP regulated genes were involved in cell cycle. Cell cycle profiling at various time points of estrogen treatment reveals that under E6-AP knockdown conditions, breast cancer cells progress slowly through S phase and eventually fail to proliferate. Knockdown of E6-AP has no effect on ovarian and uterine cells, suggesting that E6-AP has cell specific roles. Our analysis suggests that knockdown of E6-AP reduces the levels of early (C-Myc and Cyclin-D1), mid (E2F1, E2F2 and E2F7) and late (BUB1, BUBR1, MAD2, NDC80, NUF2 and CASC5) estrogen-dependent cell cycle genes. Overall our data indicate that E6-AP is a major regulator of cell cycle in breast cancer cells. E6-AP also acts as a coactivator for androgen receptor (AR) and we studied the role of E6-AP in prostate gland development. We report the generation of transgenic mice which specifically over expresses E6-AP in the prostate gland. Prostate glands in these mice are larger when compared with its wild-type litter mates, corroborating our observations that knockout of E6-AP in mice leads to impaired prostate gland development. E6-AP transgenic mice also develop prostatic intra epithelial neoplasia after 18 months of age. In addition to these observations, we also show that over expression of E6-AP in the prostate gland leads to increased Akt signaling. In order to understand the mechanism by which E6-AP regulates prostate gland growth, we generated LNCaP cells that stably overexpress E6-AP protein. Data from these cell lines show that the levels of phosphatidylinositol 3-kinase, total Akt, phosphorylated Akt (active Akt) and its down-stream target protein, GSKβ are elevated, suggesting that E6-AP regulates the PI3K-Akt signaling pathway. We further show that E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased proliferation. Overall our data suggests that E6-AP regulates the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and tumorigenesis.

Experimental autoimmune encephalomyelitis (EAE) models multiple sclerosis (MS) and is characterized by marked mononuclear cell influx in the brain. Inflammation leads to demyelinating lesions and disease. Several studies have demonstrated a role for chemokines during EAE. It remains to be determined whether these mediators modulate EAE primarily by mediating leukocyte influx into the CNS or by modifying lymphocyte activation and/or trafficking into lymphoid organs. After induction of EAE with MOG35-55, leukocyte recruitment peaked on day 14 and correlated with symptom onset, TNF-á production and production of CCL2 and CCL5. Levels of CXCL-10 and CCL3 were not different from control animals. We demonstrated that leukocyte rolling and adhesion also peaked at day 14. The treatment with anti-CCL2 or anti-CCL5 antibodies just prior to the intravital microscopy prevented leukocyte adhesion, but not rolling. To confirm the role of CCL2 on leukocyte recruitment, we performed different treatments with the mutant protein P8A, described as an inhibitor of CCL2 activity. P8A was able to decrease leukocyte adhesion and ameliorated the clinical course of disease. Our data suggest that induction of leukocyte adhesion to the brain microvasculature is an important mechanism by which CCL2 and CCL5 participate in the pathogenesis of EAE. Next, we investigated the role of kinins in driving EAE and chemokine production in the brain. The kinins are relevant mediators of inflammation and act through stimulation of two receptor subtypes, B1 and B2. For the present study B1- deficient (B1-/-), B2-deficient (B2- /-) and wild type (WT) mice were used. The incidence of disease was similar in the groups, but the disease in B2-/- mice was less severe. At day 14 after EAE induction, there was a significant decrease in the number of adherent leukocytes when compared with WT mice. EAE induced an increase of B1 mRNA in brain tissue and this was partially prevented in B2-/- mice. Expression of CCL5 was suppressed in both B1 -/- and B2 -/- mice, but CCL2 expression was only inhibited in B2-/- mice. Altogether, our results suggest that B2 receptors have two major effects in the control of EAE severity: B2 regulates the expression of chemokines, including CCL2 and CCL5, the expression of B1 receptors, and B1-dependent leukocyte influx. Blockade of chemokine action (eg. by using P8A) or production (eg. by using bradykinin antagonists) may represent a valid strategy to prevent leukocyte influx into the brain and disease severity in patients with MS.
A Encefalomielite Autoimune Experimental (EAE), modelo animal da Esclerose Multipla (EM), e caracterizada como uma doenca inflamatoria cronica, com um intenso influxo de celulas mononucleares para o sistema nervoso central (SNC). Varios estudos tem demonstrado uma participacao das quimiocinas durante a EAE, mas ainda nao foi esclarecido como esses mediadores regulam o recrutamento de leucocitos para o SNC. Apos a inducao da EAE com MOG35-55, o recrutamento de leucocitos foi marcante no dia 14 apos a inducao, com um aumento dos eventos de rolamento e adesao, correlacionando com o pico de manifestacao clinica e inflamacao, assim como um aumento da producao de TNF¿, CCL2 e CCL5. Nenhuma diferenca foi observada quanto aos niveis de CCL3 e CXCL10. O tratamento com anticorpos anti-CCL2 e anti-CCL5 2h antes da microscopia intravital foi capaz de promover uma diminuicao no processo de adesao, sem alterar o rolamento. Para confirmar o papel da quimiocina CCL2 no recrutamento de leucocitos, foram realizados diferentes tratamentos com a proteina mutante P8A, descrita como inibidora da atividade da CCL2. A proteina P8A foi capaz de diminuir a adesao de leucocitos e exibir um quadro clinico menos grave. Esses dados sugerem que a inducao da adesao de leucocitos na microvasculatura cerebral e um evento importante na patogenia da EAE e que as quimiocinas CCL2 e CCL5 podem modular este processo. Depois foi investigado o papel das cininas no recrutamento de leucocitos para o SNC no mesmo modelo. As cininas sao mediadores relevantes da inflamacao e atuam atraves de dois receptores especificos acoplados a proteina G, B1 e B2. Para este estudo foram utilizados animais WT, B1-/- e B2-/-. A incidencia da doenca foi similar entre os grupos, mas os animais B2 -/- exibiram menor gravidade da doenca. No dia 14o apos a inducao, houve uma diminuicao do numero de leucocitos aderidos nos animais knockout quando comparados com animais WT. Alem disso, animais com EAE demonstraram um aumento da expressao de receptores B1 no extrato de cerebro, parcialmente diminuidos nos animais B2 -/-. A producao de CCL5 foi diminuida nos animais B1 -/- e B2 -/-, mas CCL2 apenas foi diminuida nos animais B2 -/-. Contudo, esses resultados sugerem que receptores B2 possuem um importante papel no controle da doenca, via regulacao da expressao de B1, modulacao da quimiocina CCL2 e, consequente, controle da adesao de leucocitos. Logo, o bloqueio da atividade da quimiocina CCL2 pelo uso da P8A ou por antagonistas de BK pode representar uma importante estrategia para suprimir o influxo de leucocitos para o SNC e, consequentemente, controlar a gravidade da doenca.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
O câncer de mama é a neoplasia que mais acomete mulheres no mundo. Dentre os fatores prognósticos levados em consideração estão os receptores hormonais. O receptor de estrógeno beta (ERβ) é um dos receptores hormonais que pode ser encontrado na mama, mas que até então não é utilizado como um marcador preditivo de prognóstico tumoral por conta da sua ação paradoxal. Células tumorais de mama da linhagem MCF-7 foram utilizadas neste trabalho para averiguar a função dicotômica do ERβ no processo tumoral. De tal forma que foi avaliado alguns dos processos que são observados nas etapas do desenvolvimento do câncer de mama, como proliferação, transição epitélio-mesênquima (EMT) e investigação das células -tronco cancerosas (CSCs) . Para isso, foram utilizados o agonista de ERβ, Diarilpropionitrilo (DPN), o agonista ambíguo (ERα e ERβ) estradiol (E2) e o fator de crescimento transformante beta (TGF-β), indutor de EMT. As células que receberam tratamento com DPN obtiveram maior número de CSCs comparadas às que não foram cultivadas com esse agonista, resultado encontrado pela técnica de citometria de fluxo. As células que tiveram um tratamento prévio com TGF-β demonstraram menor taxa de proliferação e aumento na expressão de p21, uma proteína com ação bloqueadora de ciclina D1, cuja expressão ficou inalterada nos tratamentos com TGF-β e agonista de ERβ. A respeito dos genes relacionados a EMT (SLUG, SNAIL,VIMENTINA, ZEB1,TWIST1,RBFOX2,CICLINAD1 e p21), ERβ inibiu a expressão dos mesmos, sugerindo que esse receptor induza o fenômeno denominado transição mesenquimal epitelial (MET). Nesse cenário, DPN causou a diminuição da expressão de SLUG, SNAIL, TWIST, contrastando com a expressão obtida no tratamento com TGF-β que, além desses genes, também demonstrou aumento na expressão de ZEB1, RBFOX2 e vimentina. O efeito do TGF-β na EMT foi revertido ao associá-lo com DPN. Os dados corroboram com a literatura acerca do possível papel pró tumoral de ERβ no aumento da proliferação celular e da geração das células-tronco cancerígenas de mama (BSCs), além de ir ao encontro do fenótipo relacionado a MET nos tratamentos com DPN sozinho ou associado ao TGF-β. Com esses resultados, torna-se claro no nosso trabalho que dependendo o que é avaliado em relação a ação do ERβ, previamente tratado ou não com TGF-β, o seu efeito dicotômico ainda é observado.
Breast cancer is the most common neoplasm of women in the world. Among the prognostic factors taken into consideration are the hormonal receptors. The estrogen receptor beta (ERβ) is one of the hormone receptors that can be found in the breast, but is not used as a predictive marker of tumor prognosis due to its paradoxical action. Tumor cells of the MCF-7 lineage were used in this work to ascertain the dichotomic function of ERβ in the tumor process. Thus, we evaluated some of the processes that are observed in the stages of breast cancer development, such as proliferation, epithelial-mesenchymal transition (EMT) and cancer stem cell research (CSCs). For this, ERβ agonist Diarilpropionitrile (DPN), ambiguous agonist (ERα and ERβ) estradiol (E2) and transforming growth factor beta (TGF-β), inducer of EMT, were used. Cells receiving TGF- β treatment obtained a higher number of CSCs compared to those that were not cultured with this agonist, a result found by the flow cytometry technique. Cells that were pretreated with TGF-β demonstrated a lower rate of proliferation and increased expression of p21, a protein with cyclin D1 blocking action, whose expression was unchanged in TGF-β and ERβ agonist treatments. Regarding the EMT-related genes (SLUG, SNAIL, VIMENTINA, ZEB1, TWIST1, RBFOX2, CYCLINAD1 and p21), ERβ inhibited their expression, suggesting that this receptor induces the phenomenon called epithelial mesenchymal transition (MET). In this scenario, DPN caused a decrease in the expression of SLUG, SNAIL, TWIST, in contrast to TGF-β expression, which, in addition to these genes, also showed increased expression of ZEB1, RBFOX2 and VIMENTIN. The effect of TGF-β on EMT was reversed by associating it with DPN. The data corroborate with the literature about the possible ERβ pro-tumor role in increasing cell proliferation and the generation of breast cancer stem cells (BSCs), in addition to the MET related phenotype in treatments with DPN alone or associated to TGF-β. With these results, it becomes clear in our work that depending on what is evaluated in relation to the action of ERβ, previously treated or not with TGF-β, its dichotomous effect is still observed.
Dissertação (Mestrado)

SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, plays a critical role in regulation of cell signal transduction. SHP-1 is expressed not only in cells of hematopoietic lineages, but also in many non-hematopoietic cells under the control of a tissue-specific promoter, P1. In the first part of this thesis, the activity of the P1 promoter was analyzed in a region spanning 3.5 kb upstream of the major transcription start site in non-hematopoietic MCF-7 cells. An upstream Sp1 element (-126 to -118) positively regulated this TATA-box-lacking promoter. Two inverted CCAAT-elements (-332 to -328 and -66 to -62) played the important, but opposite roles, and transcription factor NF-Y predominantly bound to the two CCAAT-elements to control SHP-1 gene expression. Furthermore, incubation of MCF7 cells with 100 ng/ml trichostatin A (TSA), an inhibitor of histone deacetylase, significantly increased the activity of the P1 promoter. Mutation in the proximal CCAAT-element, however, eliminated the activating effect of trichostatin A on the promoter. In the second part of this thesis, the mechanism by which SHP-1 modulated TSA-induced MCF-7 cell apoptosis was elucidated. Analysis of cell survival signaling pathways revealed that overexpression of SHP-1 inactivated Akt ( eg. diminished phosphorylation resulted from modulation of PI3K expression) and increased caspase-9 and caspase-7 activities. Interestingly, a parallel decrease was observed in the phosphorylation of the pro-apoptosic Bcl-2 family member Bad at Ser112 as well as in the stress-activated MAP kinase JNK, both of which have been implicated in Akt- as well as ERK1/2-mediated functions. It was not surprising, therefore, to detect a diminished level of phosphorylated ERK1/2 in SHP-1-overexpressiog cells and that this effect was exacerbated by TSA treatment. Taken together, the data presented in this thesis suggest that SHP-1 expression is regulated by Sp1 and NF-Y factors, and SHP-1 sensitizes MCF-7 cells to TS

This project utilises innovative methodology to evaluate the suitability of novel three-dimensional (3D) cell culture models for investigating anti-cancer drug activity. 3D cell culture methodology was utilised as this in vitro approach is considered to recapitulate the in vivo conditions more accurately than two-dimensional (2D) monolayer cell culture. Two separate 3D cell culture model formats were developed which are amenable to automated liquid handling systems, and a variety of instruments for total well fluorescence and confocal imaging. The first 3D cell culture assay developed was in a 384-well format, and was validated as suitable for use for drug discovery. The second 3D cell culture assay was optimised for 1536-well format, specifically created for extensive drug combination studies. The 3D breast cancer cell culture assay developed for drug discovery utilised the breast cancer cell lines of MDA-MB-231 (endocrine receptor- and ErbB2 receptor-negative), MCF-7 (endocrine receptor-positive) and BT-474 (ErBb2 receptor over-expression). This 3D cell culture assay was miniaturised to a 384-well format, developed to be semi-automated and was thoroughly characterised for sensitivity and reproducibility. In addition, measurements of metabolic activity or spheroid morphology can be utilised for determination of drug activity. To validate the 3D breast cancer cell culture assay for use in high-throughput applications, a pilot screen comprising of 741 clinically relevant drugs was completed. Results from the pilot screen identified a number of drugs with anti-breast cancer activity that warranted further investigation. The drugs of interest were a mixture of drugs with both novel and demonstrated anti-cancer activity.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text

Thesis (Ph. D.)--West Virginia University, 2004.
Title from document title page. Document formatted into pages; contains x, 160 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

La obesidad es uno de los factores más reconocidos en el desarrollo del cáncer de mama, y el tejido adiposo juega un papel muy importante en esta patología debida la secreción de sus diversas citosinas, principalmente Leptina y Adiponectina. La base molecular de esta asociación no es bien conocida y muy pocos son los que han estudiado a los receptores de Adiponectina (AdipoR1 y AdipoR2) y la influencia de la Leptina sobre ellos. Por lo que se requiere elucidar los mecanismos por los cuales niveles altos de Leptina en el organismo generan un aumento en el riesgo de desarrollo de cáncer de mama y si esta proteína tiene una influencia sobre AdipoR1 y AdipoR2, la cual podría afectar la acción anti-proliferativa de la Adiponectina.
Se ha documentado el papel que juega el tejido adiposo a través de Leptina y Adiponectina implicados en el desarrollo y progreso del cáncer de mama, pero muy pocos son los estudios sobre AdipoR1 y AdipoR2 y la influencia de la Leptina sobre ellos. Objetivo: Analizar la expresión de AdipoR1 y AdipoR2 modulada por concentracionesdiferenciales de Leptina en un modelo de obesidad asociado a cáncer de mama en las líneas celulares MCF-7, MDA-MB231 y HCC1937. Métodos: Se analizó la expresión de AdipoR1 y AdipoR2 por PCR en tiempo real utilizando sondas TaqMan®, mediado por concentraciones de Leptina (0 ng/mL, 10ng/mL, 100 ng/mL y 1000 ng/mL) en líneas celulares de cáncer de mama: MCF-7,MDA-MB231 y HCC1937. Se caracterizó cada línea celular por Inmunohistoquímica. Resultados: La Leptina generó un aumento de la población celular en MCF-7 (23.8%, 10 ng/mL, 48 h); en MDA-MB231 la población aumentó hasta un 17.02% (1000 ng/mL, 72 h) y en HCC1937 aumentó en un 17.24% (1000 ng/mL, 72h). En MCF-7 la expresión de AdipoR1 disminuyó (3.81%, 1000 ng/mL), excepto para 100 ng/mL (64.03%). Laexpresión de AdipoR2 aumentó hasta 13.74 veces (10 ng/mL) respecto al control. EnHCC1937 la expresión de AdipoR1 disminuyó hasta un 86.28% (10 ng/mL), mientras que la expresión de AdipoR2 tuvo una disminución hasta un 50.3% (100 ng/mL). Conclusiones: La concentración de normo-peso (10 ng/mL) de Leptina generó un aumento de la expresión de ambos receptores de Adiponectina.
Proyecto DSA/103.5/16/10569

Trabajo de investigación en líneas celulares de cáncer de mama en módelo de obesidad y su expresión de Receptor de leptina con diferentes concentraciones de tamoxifeno
Introducción El cáncer de mama es el principal tipo de cáncer que afecta a las mujeres a nivel mundial; es una patología que se puede desencadenar debido a múltiples factores que conllevan a una proliferación anormal y descontrolada de células que componen la glándula mamaria. Uno de los factores que ha incrementado la incidencia y presenta peor pronóstico de este tipo de cáncer es la obesidad. En esta condición son secretadas altas cantidades leptina, una proteína que presenta actividad proliferativa, mitogénica, antiapoptótica y proinflamatoria que puede resultar antagónica al tratamiento quimioterapéutico. Objetivo: El objetivo del proyecto fue identificar la modulación de la expresión génica de receptores de leptina y la proliferación celular en líneas celulares de cáncer de mama (MDA-MB 231, MCF-7 y HCC1937) como resultado de su estimulación con leptina y tamoxifeno y establecer un modelo de abordaje inicial para el estudio de los subtipos de cáncer de mama y su comportamiento a la respuesta de la acción de las adipocinas y su posible relación con el mecanismo de resistencia a quimioterapéuticos como el tamoxifeno en líneas celulares ER positivo y triple marcador negativo Material y métodos: Se realizó la evaluación de la modulación de la expresión del receptor de leptina en presencia de estímulos de leptina y tamoxifeno en las líneas celulares de cáncer de mama MCF 7, MDA MB 231 y HCC 1937 mediante ensayos de proliferación con tinción de cristal violeta, y análisis de la expresión de mRNA del receptor de leptina mediante su transcripción a cDNA, PCR en punto final y RT-PCR, así mismo se evaluó la expresión de la proteína del receptor de leptina mediante ELISA. Resultados: Se determinó que la leptina es capaz de modular positivamente la proliferación de las tres líneas celulares de cáncer de mamá y el tamoxifeno es capaz de ejercer un efecto antiproliferativo en las mismas, se identificó que la capacidad del tamoxifeno para disminuir la proliferación de células cancerígenas se ve disminuida en presencia de leptina, aunada a cambios en la modulación de la expresión de su receptor. Del mismo modo se determinó la concentración de la proteína de ObR mediante la técnica de ELISA obteniéndose una mayor concentración en todos los casos en comparación con el control, no obstante la variación no es estadísticamente significativa. Conclusiones: El tamoxifeno es un agente quimioterapéutico capaz de inducir una mayor modulación de la expresión del ObRb en las líneas celulares de cáncer de mama, lo cual puede estar relacionado con la disminución de su actividad antiproliferativa, mientras que la leptina genera un efecto proliferativo en las tres líneas celulares, lo cual corresponde con los reportes de su actividad proliferativa, antiapoptótica y mitogénica.
SEIA- UAEMéx 4563-2018 CIV

ABSTRACT MICROARRAY APPLICATIONS FOR DETERMINATION OF THE EFFECTS OF EMODIN ON BREAST CANCER CELL LINES Ekenel Qomi, Emilia M.S., Department of Biotechnology Supervisor: Prof. Dr. Mesude Iscan Co-Supervisor: Assoc. Prof. Dr. Nursen Ç
oruh February 2012, 191 pages Cancer is a genetic disease that is characterized by uncontrolled cells growth. Breast cancer is a type of cancer originating from breast tissue. Some breast cancers are sensitive to hormones such as estrogen which makes it possible to treat them by blocking the effects of these hormones in the target tissues. These require less aggressive treatment than hormone negative cancers. Breast cancers without hormone receptors, are higher-risk, and are treated more aggressively. The aim of our study is to investigate the effect of emodin on MCF-7 which is ER (estrogen receptor) positive, and MDA-MB-231 (ER negative) cancerous cell lines. Emodin which is a phytoestrogen component, extracted from rheum (genus) plant, has been reported to suppress the growth of tumor in some clinical situation, and it&rsquo
s found that emodin induced apoptosis through the decrease of Bcl-2/Bax ratio and the increase of cytoplasm cytochrome c concentration in human breast cancer Bcap-37 cells. Comparing the effect of emodin between ER positive and ER negative cells at the molecular level was investigated by Microarray analysis of gene expressions using Affymetrix Human Genome U133 plus 2.0 Array. The microarray data analysis was performed by using BRB-Array Tools, v.4.2.0. GST and its classes
Alpha, Mu, Pi, Theta, Sigma, Omega, Zeta and Kappa is our interested genes because of its role in regulating susceptibility to cancer, by their ability to metabolize reactive electrophilic intermediates to usually less reactive and more water soluble glutathione conjugates. And also its have a role in detoxifying the damage caused by oxidative stress which is a result of the radiotherapy. v The differentially expressed genes from emodin treated and untreated control breast cancer cell lines were compared after normalization and filtering and annotated, it was shown that the top 10 highly (significantly) varied genes belong to the biological processes such as (namely) cell cycle, cell division, cell proliferation, mitosis and meiosis, this insure the relation of emodin to the cell growth processes in the cancerous cells. The analysis of the change on the cell growth confirmed the anti-tumor effect of emodin. About the effect of emodin treatment on MCF-7 and MDA-MB-231 cancerous cell lines separately
Both cells its significant genes was belong to cell growth biological processes, in MCF-7 cells in-addition other biological processes was shown, for example
stimulus to estradoil response, and the metabolism of xenobiotic by cytochrome p450, so CYP1A1 gene code for a protein which is used in emodin metabolism. The varied gene number was nearly 4400 gene from the scatter plot result in MCF-7 cells while in MDA-MB-231 cells it was nearly 3400 gene, these result insured the effect of emodin as a phytoestrogenic component as MCF-7 cells are ER positive cells, so emodin bind to the ER in MCF-7 cells and affected more gene number than MDA-MB-231. More number of GST enzyme classes changed in MCF-7 cells than MDA-MB-231, and the effect of emodin as anti-cancer showed different change of GST genes between MCF-7 and MDA-MB-231. The results confirmed by network analysis done, to find the most related genes to our top 10 regulated gene list, and these genes were analyzed
most of them where in our gene list, and their regulation after emodin treatment analyzed and the result was supported to emodin as anti-tumor and phytoestrogenic component.

La protéine kinase D1, PKD1, est une nouvelle sérine/thréonine kinase activée par de nombreux mitogènes et dérégulée dans de nombreux types de cancers dont le cancer du sein, ce qui suggère un rôle de cette kinase dans la prolifération cellulaire et la tumorigenèse. Cependant, le rôle précis et les cibles de PKD1 ne sont pas encore bien connus. Au cours de ce travail, nous avons tout d’abord démontré que PKD1 est activée par les facteurs de croissance épidermique (EGF) et fibroblastique (FGF) et qu’elle régule la voie de signalisation de l’insulin-like Growth Factor-I (IGF-I). D’autre part, nos résultats démontrent que PKD1 favorise les propriétés pro-prolifératives et pro-tumorales des cellules MCF-7 dérivées d’un adénocarcinome mammaire humain estrogéno-dépendant. Ces mécanismes mettent en jeu des voies de signalisation dépendantes de protéines kinases (la voie MEK/ERK) et hormonales (la voie estrogène/REα). Ainsi, l’ensemble de ce travail fait apparaître PKD1 comme une nouvelle cible thérapeutique anti-tumorale potentielle
Protein kinase D1, PKD1, is a novel serine/threonine kinase which can be activated by mitogens and whose expression is altered in many tumors such as breast cancer, suggesting a role for this kinase in cancer development. However, its precise role and targets are still unclear. Our study identified PKD1 as a new regulatory kinase implicated in the control of IGF-I signal transduction pathway. Furthermore, we showed that PKD1 enhances estrogen-dependent MCF-7 breast cancer cell proliferation and tumorigenesis through the regulation of MEK/ERK and estrogen/ERα pathways. Thus, this work may define PKD1 as a novel potential anti-tumor therapeutic target

Magister Scientiae (Medical Bioscience) - MSc(MBS)
This study investigated the effects of TKIs on the growth and proliferation of MCF-7 breast carcinoma cells in culture. MCF-7 cells were exposed to different concentrations of TKIs alone and in combination with each other. Inhibition of cell growth by TKIs used individually occurred in a dose- and time-dependent manner. When EGFR Inhibitor I, EGFR Inhibitor II/BIBX1382 and the multi-specific EGFR/ErbB-2/ErB-4 Inhibitor were used in combination with each other at equimolar log dose concentrations, the combined effects on cell growth was significantly different to inhibitors used individually as reflected in a decreased EC50 (IC50) during combination treatments. Generally, for the combinations with DOX, CPL and the TKIs, synergistic as well as antagonistic effects were observed at isoeffective concentrations with resultant decreases in dose reduction indices (DRIs) implying greater efficacies with the respective combinations. In this study, conventional PCR was used to detect and illustrate the presence of the EGFR gene in the samples, while RT-qPCR was used to determine the mRNA expression levels of this gene in MCF-7 breast carcinoma cells

碩士
國防醫學院
生物及解剖學研究所
93
Several frog oocyte-derived antitumoral ribonucleases are noted these days, such as onconase from Rana pipiens and RC-RNase from Rana catesbeiana. RC-RNase belongs to the pancreatic ribonuclease A superfamily, is a pyrimidine-guanine sequence specific ribonuclease, which shares 48.2％ amino acid sequence homology to onconase, which is a well-known anti-tumor drug and currently on its phase III trail in USA. RC-RNase processes cytotoxicity for varies tumor cells but rarely to normal cells such as fibroblasts. Our present studies demonstrated that RC-RNase is toxic to estrogen receptor (ER)-positive breast cancer cells, MCF-7 and ZR-75-1, but not to ER-negative ZR-75-30 and MDA-MB-231 cells. RC-RNase induced cell death through an apoptotic pathway, and blocked the expressions of ER proteins, Bcl-2 and Bcl-XL. We also found that the amount of ER mRNA expression accumulated in MCF-7 cells with a time- and dose-dependent conditions. The ER protein expression in a selected RC-RNase-resistant MCF-7 cell line was higher, but, its mRNA level was uninfluenced. These results inferred that RC-RNase induced cytotoxicity of MCF-7 cells could probably act on the translation level to block the ER protein expression, which then is not able to keep the cells alive. The results that RC-RNase activates caspase-3-like, caspase-8 and caspase-9 in MCF-7 cells indicated that mitochondria-mediated apoptosis mechanism could be involved. Over-expression of ER in MCF-7 cells by transfection of a plasmid, pCMV-sER-neo, rescued RC-RNase induced cytotoxicity. We concluded that RC-RNase-induced cytotoxicity in MCF-7 cells is modulated by ER.

The pineal hormone, melatonin, inhibits tumorigenesis and functions as an oncostatic and antiproliferative agent in endocrine responsive breast tumors. Physiological concentrations of melatonin have been shown to inhibit the proliferation of estrogen receptor (ER)-positive MCF-7 human breast cancer cells and to modulate the expression of both ER mRNA and protein. Melatonin inhibition of MCF-7 cell proliferation is serum dependent, indicating an interaction between melatonin and serum components. To examine the effects of melatonin on ER activity, ER transactivation assays were performed using an estrogen response element (ERE)-luciferase reporter construct transiently transfected into MCF-7 cells. Pre-treatment of MCF-7 cells with melatonin for as little as 5 min followed by either epidermal growth factor (EGF) or insulin resulted in the estrogen independent transactivation of the ER. This effect appears to depend on the sequential treatment with melatonin and EGF or insulin, since none of these compounds individually transactivated the ER. This modulation of ER transactivation was associated with changes in ER phosphorylation levels; treatment with melatonin followed by either EGF or insulin increased phosphorylation of the ER, however, melatonin alone decreased basal ER phosphorylation. Here we report that the membrane bound G-protein coupled melatonin receptor, Mel$\sb{\rm 1a}$, is expressed in MCF-7 cells. Further, melatonin modulation of ER transactivation was blocked by pertussis toxin, a G$\sb{\rm\alpha i}$-protein coupled receptor inhibitor, suggesting that the ligand independent transactivation of the ER in MCF-7 cells could result from crosstalk between the G-protein coupled melatonin receptor pathway and the EGF/insulin tyrosine kinase receptor pathways
acase@tulane.edu

碩士
國立臺灣師範大學
生命科學研究所
93
Inorganic arsenics, the common environmental pollutants, are widely dispersed and the well-documented human carcinogens associated with cancers of skin, lung and bladder and can induce cytotoxicity, chromosomal abnormalities, oxidative stress, altered DNA repair, altered growth factors etc. Several reports indicated that inorganic arsenic might somehow act through an estrogenic mode of action. In this study, effects of sodium arsenite and sodium arsenate on cytotoxicity, cell proliferation and the expression of estrogen receptorα(ERα) in breast adenocarcinoma MCF-7 cells are determinated. The preliminary results show that the inhibiting threshold concentrations of sodium arsenite and sodium arsenate on cell growth are at 1μM and 10μM, respectively, whereas their concentrations lower than inhibiting threshold concentrations promoted cell growth instead of cell toxicity. Time course studies on cells treated with 0.1~1μM sodium arsenite and 1~10μM sodium arsenate for 24 h, and observed consecutive six days without drugs, MCF-7 cells proliferation exhibited continuous increase. Cotreated with drugs and estrogen antagoist (ICI 182,780), MCF-7 cell proliferations were completely inhibited. Data of Western blotting show that the expression of ERα were decreased in cells treated with low dose of sodium arsenite and sodium arsenate, but expression of estrogen receptorβ were not affected. Immunocytochemical studies with fluorescent microscopy illustrated that ERα displayed a diffusion distribution in cytoplasm and were more concentrated within nucleus of control cell and E2-treated cells. In arsenic-treated cells, the ERαonly displayed in cytoplasm. Whole-cell competitive estrogen-receptor binding assay demonstrate that 0.1, 0.5 and 1μM sodium arsenite could bound with 28%, 35% and 48% in ER respectively. In this study it was concluded that arsenite could bind with estrogen receptor. Based on results above, MCF-7 cells treated with a low dose sodium arsenite and sodium arsenate affect cell proliferation, ERαexpression, and competition of estrogen receptor binding；and indicate that inorganic arsenics do have the characteristics of environmental hormone for inducing physiological effect, through a estrogen model.

碩士
臺北醫學大學
醫學研究所
97
Breast cancer is prevalent among women and estrogens are involved in two-thirds of breast tumors. Estrogens bind with estrogen receptors (ERs) in the cytoplasm, and are translocated to nucleus as transcription factors to activate the expression of a number growth factors and promote tumor growth. Up till now, two ERs have been identified: Estrogen receptor α (ERα) and β (ERβ). The role of ERα in the estrogen-mediated pathway has been carefully studied, whereas the function of ERβ is still unclear. Previous studied have demonstrated that the expression of cytoplasmic estrogen receptor β (ERβ) indicates the poor survival and prognosis. ERβ has also been found to translocate to mitochondria, but its mechanism remains unclear. We hypothesize that ERβ agonists can attenuate mitochondrial biogenesis, suppress mitochondrial respiration, inhibit ATP production, enhance glycolysis and lactate accumulation, and promote tumor proliferation in breast cancer. In this study, MCF-7 breast cancer line was treated with ERβ agonists, diarylpropionitrile (DPN) and 17β-estradiol (E2), and the expression of major ERβ was detected in mitochondria of MCF-7. E2 and DPN were found to promote cell growth. In cell proliferation MTS assay, E2 or DPN could enhance cell proliferation by 2.6 and 1.6 times, respectively. In addition, E2 or DPN were found to downregulate mitochondrial ND-1 mRNA level to 76.7% and 76.4%, respectively. The expressions of respiratory complex proteins were also decreased by DPN or E2 treatment. Moreover, the effect of E2 and DPN on mitochondrial membrane potential was assessed. DPN attenuated mitochondrial membrane potential by 24.8% and 13.5% by E2, as compared with the control group. In assay for the reactive oxygen species (ROS), we found that DPN and E2 could reduce the oligomycin-induced ROS generation. A 9.8% decrease in ATP production by E2 and 9.5% decrease by DPN were also found. Furthermore, hypoxia-inducible factor 1α (HIF1-α) and pyruvate dehydrogenase kinase (PDK) protein levels were increased by DPN or E2, but the expression of pyruvate dehydrogenase (PDH) was decreased. The activities of both glycolytic enzyme phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were increased, indicating that the means the cell relied more on glycolysis for supply of energy. By use of ERβ shRNA, the LDH activity was reduced by DPN or E2 treatment. In conclusion, we demonstrated that ERβ agonists inhibit mitochondrial function and cancer cells therefore derive most of their energy from anaerbolic glycolysis to maintain cell growth. Key words：breast cancer、estrogen receptor β (ERβ)、mitochondria

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

O cancro da mama permanece o cancro mais predominante na mulher e estima-se que a mortalidade aumente 43% até 2030 em todo o mundo. Aproximadamente 70% dos tumores mamários são positivos para o recetor de estrogénio (ER), tornando possível o tratamento com terapia hormonal. O cancro da mama é uma doença heterogénea com múltiplos fatores associados à sua origem e comportamento invasivo, e apesar dos avanços científicos, a resistência à terapia continua a ser um assunto na ordem do dia. Este projeto procurou investigar como é que a atividade do ER pode mudar na presença de diversos fatores e a relação com a resistência à terapia endócrina. Primeiramente testámos por imunocitoquímica uma combinação de anticorpos que permitiu avaliar a interação do ER com dois co-ativadores, PPARγ e PGC-1β. Os testes realizados sugerem a existência de co-localização nas células MCF-7 resistentes ao tratamento com tamoxifeno, o que poderá resultar numa ativação ligando-independente do ER e resistência endócrina. Depois fomos investigar o efeito do inibidor da metiltransferase SETD7, (R)-PFI-2 só ou em combinação com 17β-estradiol (E2), no ciclo celular e na proliferação, em células MCF-7 e T-47D. Observamos que o tratamento com (R)-PFI-2 pareceu induzir paragem do ciclo em G0/G1 em ambas as linhas celulares. No entanto, a combinação (R)-PFI-2 + E2 não inibiu o aumento do ciclo celular provocado pelo E2. Nas mesmas células quisemos também ver o efeito do inibidor na expressão de ER e um dos seus genes alvo, o recetor de progesterona (PR). Os nossos resultados indicam que não houve estimulação da expressão de PR. Estes resultados vão de encontro ao descrito na literatura, que indica que a atividade de SETD7 é necessária para o recrutamento eficiente de ER para os promotores dos genes alvo e consequentemente para ativar a transcrição. Portanto, não encontramos uma correlação entre os efeitos da inibição da SETD7 na expressão de PR e no ciclo celular estimulado por E2. Além disso fomos observar o efeito dos antagonistas 4-OH-Tamoxifeno e ICI 182 780 quando combinados com (R)-PFI-2. Nas células MCF-7 a proliferação foi inibida na presença destes compostos, mas quando juntamos (R)-PFI-2 não houve um efeito aditivo inibitório. Por outro lado, nas células T-47D não observamos efeito dos antagonistas, o que indica resistência à terapia, mas quando combinados com (R)-PFI-2, este foi capaz de reduzir a resistência endócrina demonstrada por estas células. Mais estudos são necessários de forma a confirmar estes resultados e o papel da SETD7 no cancro da mama. No entanto, propormos que a inibição de SETD7 em células resistentes aos antagonistas estudados pode ser uma estratégia para diminuir a resistência.
Breast cancer remains the most prevalent cancer in women and its mortality is estimated to increase 43% until 2030 worldwide. Approximately 70% of breast cancers are positive for the estrogen receptor (ER), making it possible to treat them with hormone therapy. Breast cancer is a heterogeneous disease with multiple factors associated with its origin and invasive behavior, and despite scientific advances, resistance to therapy remains a major issue. This project sought to investigate how ER activity can change in the presence of several factors and the relationship with resistance to endocrine therapy. Firstly, we tested for immunocytochemistry a combination of antibodies that allowed us to evaluate the interaction of ER with two co-activators, PPARγ and PGC-1β. The performed tests suggest co-localization in MCF-7 cells resistant to tamoxifen treatment, which may result in a ligand-independent activation of ER and endocrine resistance. We then investigated the effect of the methyltransferase inhibitor SETD7, (R)-PFI-2 alone or in combination with 17β-estradiol (E2) on the cell cycle and proliferation in MCF-7 and T-47D cells. We observed that treatment with (R)-PFI-2 appeared to induce a G0/G1 cell cycle arrest in both cell lines. However, the (R)-PFI-2 + E2 combination did not inhibit E2 cell cycle enhancement. In the same cells, we also wanted to see the effect of the inhibitor on ER expression and one of its target genes, the progesterone receptor (PR). Our results indicate that there was no increase in the expression of PR. These results are in agreement with the literature, which indicates that the activity of SETD7 is necessary for the efficient recruitment of ER to the promoters of the target genes and consequently to activate the transcription. Therefore, we did not find a correlation between the effects of the SETD7 inhibitor on ER-mediated expression of PR and E2-stimulated cell cycle. In addition, we investigated the effect of 4-OH-Tamoxifen and ICI 182 780 antagonists when combined with (R)-PFI-2. In MCF-7 cells proliferation was inhibited in the presence of these compounds, but when (R)-PFI-2 was joined there was no inhibitory additive effect. On the other hand, in T-47D cells no effect of the antagonists was observed, indicating resistance to therapy, but when combined with (R)-PFI-2, the antagonists were able to reduce the endocrine resistance demonstrated by these cells. Further studies are needed to confirm these findings and the role of SETD7 in breast cancer. However, we propose that the inhibition of SETD7 in cells resistant to the antagonists studied could be a strategy to decrease resistance.
Mestrado em Biomedicina Molecular

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

The pineal hormone, melatonin has repeatedly been shown to inhibit the proliferation of estrogen receptor alpha (ERalpha)-positive MCF-7 human breast cancer cells. Previous reports have suggested that the actions of melatonin can be mediated either through G protein-coupled membrane receptors, or via retinoid orphan receptors (RORalpha), which constitutively activate gene transcription through their corresponding response elements termed ROREs. Transient transfection assays using an RORE-luciferase reporter construct have shown that the endogenous (RORalpha), receptors expressed in MCF-7 breast cancer cells exhibited a high basal level of transcriptional activity, which was further stimulated by serum. In the presence of serum, both the transcriptional activity and DNA binding ability of (RORalpha), were repressed by melatonin treatment, even though melatonin had no effect on RORalpha protein levels. Although RORalphas were originally proposed as nuclear receptors for melatonin, the direct binding of melatonin to these receptors can not be repeated. Therefore, it is not clear how melatonin can modulate (RORalpha), functions in MCF-7 cells. Consistent with the results from other systems, RORalpha transcriptional activity in MCF-7 cells was found to be responsive to intracellular calcium concentration ([Ca2+]i), calmodulin (CaM), and Ca2+/CaM-dependent protein kinases. Meanwhile, melatonin not only affected CaM subcellular distribution, but also modulated [Ca2+]i change induced by another known calcium stimulator, ATP, indicating that melatonin may regulate RORalpha transcriptional activity via its modulation of the Ca2+/CaM signaling pathway in MCF-7 cells. In exploring the biological functions of (RORalpha), receptors, we found that the decrease in RORalpha protein levels by an (RORalpha), antisense oligonucleotide con-elated with growth inhibition in MCF-7 cells. Our data suggest that RORalpha receptors may play a role in stimulating the proliferation of MCF-7 cells, and thus, the repressive effect of melatonin on these receptors may, at least in part, mediates melatonin's antiproliferative effects on breast cancer cells
acase@tulane.edu

Melatonin, the hormonal product of the pineal gland, has been shown to inhibit the development of mammary tumors in vivo and the proliferation of MCF-7 cells in vitro by mechanisms not yet identified. Previous studies have demonstrated that only human breast cancer cells which express estrogen receptor (ER) are responsive to the antimitogenic effects of melatonin, suggesting a link between the effects of melatonin and the estrogen-response pathway. MCF-7 human breast cancer cells, which are ER-positive and responsive to the mitogenic actions of estrogen, were used to examine the possible association between the antiproliferative activity of melatonin and its ability to modulate the estrogen-response pathway at the level of ER expression. In order to determine the mechanism(s) by which melatonin regulates ER expression in MCF-7 cells, the relationship between ER protein expression, estrogen binding activity, steady-state levels of ER mRNA, the level of ER gene transcription, and the stability of the ER transcript were examined in response to melatonin Physiologic concentrations of melatonin significantly decreased estrogen binding activity, expression of immunoreactive ER protein, and steady-state levels of ER mRNA in a dose-specific and time-dependent manner in both complete and estrogen-depleted media. However, melatonin did not alter receptor affinity and was unable to compete with estrogen for binding to the ER. Studies in a yeast transcriptional assay system confirmed that melatonin does not directly bind to the ER to modulate ER expression. The decreased expression of ER protein in response to melatonin appears to be directly related to both suppressed ER gene transcription and decreased half-life of the ER message. This regulation is independent of the synthesis of new proteins since cycloheximide did not block the melatonin-induced decrease in ER mRNA. The expression of several estrogen-induced genes were differentially regulated by melatonin, suggestive of the interplay of early versus late events in the action of melatonin. Thus, it appears that the antiproliferative actions of melatonin may be mediated, at least in part, through the suppression of the estrogen-response pathway of MCF-7 cells
acase@tulane.edu

碩士
國立陽明大學
傳統醫藥學研究所
96
The aim of this study was to correlate the role of platelet-derived growth factor receptor (PDGFR) with clinicopathological manifestations in Asian female with breast cancer and in Si-Wu-Tang (SWT)-treated MCF-7 breast cancer cell line. Breast cancer specimens were obtained from patients receiving breast surgery in Taipei-Veterans General Hospital from Jan. 2001 to Dec. 2003. The expressions of PDGFR-α and β in breast tumors were performed by using immunostaining with antibodies against PDGF receptors, followed by correlating the PDGFR expression levels with clinicopathological manifestations. Besides, the effects of SWT extract on PDGFR-β gene expression in MCF-7 cells were evaluated by immunohistochemistry, flow cytometry, RT-PCR and quantitative PCR. The results showed that high expression of PDGFR-β, but not PDGFR-��, significantly correlated with larger tumor size, lymph node metastasis and lymphovascular invasion, while no significant difference was found between PDGFR-β and hormone receptors or HER2/neu status. Multivariate analysis revealed that overexpression of PDGFR-β was a significant prognostic factor in predicting overall survival with a hazard ratio of 4.264. Repeated administration of SWT extract induced MCF-7 cell growth and the expression of PDGFR-β, both in gene expression levels and cell migration assay. We conclude that the expression of PDGFR-β correlates to the invasiveness of tumor rather than tumor characteristics in Asian female with breast cancer and SWT might increase the gene expression of PDGFR-β in MCF-7 breast cancer cells.

We have used a novel estrogen receptor-(ER) negative, mixed basal/luminal, aggressive cell line, TMX2-28, as a model to study breast cancer. cDNA microarray comparison of TMX2-28 and its parent non-aggressive, ER-positive cell line, MCF-7, identified 1402 differentially expressed transcripts. Two-hundred upregulated transcripts were sorted by biological function and the expression of selected genes was assessed in TMX2-28 cells, MCF-7 cells, non-tumorigenic human mammary epithelial cells (HMECs), and thirty frozen human breast carcinoma specimens using real time RT-PCR. Four genes were selected for further studies: phospholipase D1 (PLD1), mitogen-inducible gene 2 (MIG2), S-phase kinase-associated protein 2 (SKP2), and paralemmin (PALM). PLD1 mRNA is expressed ten times more in TMX2-28 cells than in MCF-7 cells, and PLD1 is moderately expressed in non-tumorigenic HMEC lines 184, 184A1 and 184AA2. PLD1 mRNA levels were higher in breast tumors that expressed high mRNA levels of basal CKs 5 and/or 17. PLD1 protein was overexpressed in 10 of 42 (24%) breast tumors examined by IHC. Evaluating the expression of PLD1 with other signaling molecules, phospho-Akt and phospho-mTOR, we found that five PLD1-positive tumors were negative for phospho-Akt expression, but positive for phospho-mTOR expression. MIG2 is overexpressed 17-fold in TMX2-28 cells compared to non-aggressive MCF-7 cells and HMECs. MIG2 showed high mRNA levels in 30% of the human breast carcinoma specimens. siRNA-mediated suppression of MIG2 in TMX2-28 cells reduced TMX2-28 cell invasion by 48% compared to cells transfected with siRNAs against GAPDH. We also found that MIG2 protein was expressed in half of the breast tumors tested by IHC and showed heterogeneous expression among 21 tissues from reduction mammoplasty. SKP2 is overexpressed in TMX2-28 cells compared to MCF-7 cells by 13-fold, and SKP2 was not expressed in the HMEC lines. SKP2 mRNA levels were higher in breast tumors that expressed basal cytokeratins (CK) 5 and/or 17. Lastly, PALM is overexpressed in TMX2-28 breast cancer cells compared to MCF-7 and HMECs. We further evaluated the expression of PALM in human breast carcinomas and found that PALM is highly expressed on the plasma membrane of cells in roughly half of the breast tumors.

碩士
臺北醫學大學
醫學技術學系
95
The purpose of this investigation is to study the effects of 17 beta-estradiol and nicotine on nicotinic acetylcholine receptor alpha9 ( nAChR alpha9 ) gene expression and the cross-linking signaling pathways in MCF-7 human breast cancer cells. Using physiological concentrations of 17 beta-estradiol ( 1 nM ) and nicotine ( 10 microM ) could increase nAChR alpha9 mRNA expression after 48 hours treatment. Using PI3K inhibitor, LY 294002, could block both cross-linking pathway in MCF-7. PD 98059 and SP 600 125, inhibitors of mitogen-activated protein kinase (MAPK) pathways, were also involved in both cross-linking pathways in MCF-7. Using serial deletion luciferase activity assay and site-directed mutagenesis assay to predict the most important transcription factor binding sites on nAChR alpha9 gene promoter. These results demonstrate the cross-linking pathways of 1 nM 17 beta-estradiol and 10 microM nicotine were through PI3K/Akt/p-ER alpha( Ser 167 ) and MEK1, 2/ ERK1,2/JNK1,2/p-ER alpha( Ser 118 ). The most important transcription factor binding sites on nAChR alpha9 gene promoter for 17 beta-estradiol are Vitamin D Receptor ( VDR ) and Activated Protein- 1 ( AP-1 ). The most one for nicotine is Activated Protein- 1 ( AP-1 ).

Απο τις απαρχές της μελέτης του PAR1, είχε βρεθεί οτι βρίσκεται σε στενή συνάφεια με τον καρκίνο, με ποικιλία πειραμάτων που έγιναν σε καρκινικές σειρές και σε διάφορα πειραματικά μοντέλα ζώων. Οι σκοποί της παρούσας εργασίας μπορούν να συνοψιστούν ως εξης: Η διερεύνηση της έκφρασης του υποδοχέα 1 της θρομβίνης (PAR1) σε καρκινικές σειρές προερχόμενες από ανθρώπινους όγκους και συγκεκριμένα: Στις σειρές από καρκίνο του προστάτη PC3 και LNCaP και στις σειρές από καρκίνο του μαστού MDA-231 και MCF-7. Η διερεύνηση της λειτουργικότητας του παραπάνω υποδοχέα στις προαναφερθείσες σειρές και το αποτέλεσμα της αναστολής του υποδοχέα στην επιβίωση και στον πολλαπλασιασμό των μελετώμενων κυττάρων. Η διερεύνηση της ενεργοποίησης της ΜΑΡ κινάσης μέσω του PAR1. Και τελικά, η διερεύνηση της έκφρασης του PAR-1 σε ασθενείς που χειρουργήθηκαν για καρκίνο του πνεύμονα στην Παν/μιακη Καρδιοθωρακοχειρουργική κλινική της Πάτρας, απο τον Καθηγητή Κο Δ. Δουγένη και την ομάδα του. Ο ανταγωνιστής του PAR-1, SCH 79797, προκάλεσε μείωση του κυτταρικού πολλαπλασιασμού των προαναφερθέντων καρκινικών σειρών, όπως μελετήθηκε με τη μέθοδο ΜΤΤ και την ενσωμάτωση ραδιενεργού θυμιδίνης. Αυτή η μη ειδική ανταπόκριση όλων των μελετώμενων σειρών στον SCH, αποδόθηκε κατόπιν στο γεγονός ότι αυτοί οι ανταγωνιστές δεν ήταν απολύτως εκλεκτικοί για τον PAR-1 όπως πιστευόταν, όταν σχεδιάστηκαν. Ο συγκεκριμένος ανταγωνιστής επιλέχθηκε μεταξύ των λίγων, της μοναδικής κατηγορίας που υπήρχε, όταν ξεκίνησαν τα πειράματα. Η θρομβίνη υπερδιπλασίασε τον πολλαπλασιασμό της σειράς PC3 και δεν είχε κανένα αποτέλεσμα στον πολλαπλασιασμό της σειράς MDA-231. Το τελευταίο συμφωνεί και με προηγούμενη έρευνα όπου καταδείχθηκε ότι η θρομβίνη δεν επηρεάζει τον πολλαπλασιασμό, αλλά μειώνει τη μεταστατικότητα της σειράς MDA-231 (Kamath et al., 2001). Η αύξηση του πολλαπλασιασμού των καρκινικών κυττάρων του προστάτη PC3, από τη θρομβίνη γίνεται μέσω ενεργοποίησης του υποδοχέα της PAR-1, και μέσω ενεργοποίησης της ΜΑΡ κινάσης, όπως φάνηκε από τα πειράματα στα οποία χρησιμοποιήθηκε ο ειδικός αγωνιστής του PAR1, το εξαπεπτίδιο SFLLRN. Από κάποια πρώτα ενδεικτικά πειράματα φαίνεται ότι η ενεργοποίηση της ΜΑΡΚ λαμβάνει χώρα μέσω διενεργοποίησης του EGFR, κάτι που έχει αποδειχθεί για άλλους GPCRs. Η έκφραση του PAR1 όπως μελετήθηκε με RT-PCR, σε δείγματα ασθενών που χειρουργήθηκαν για κακοήθεις όγκους στους πνεύμονες, ανιχνεύθηκε σε όλα τα δείγματα καρκινικού ιστού. Η υψηλότερη έκφραση του PAR1 ανιχνεύθηκε στον ασθενή με μελάνωμα και στον ασθενή του υψηλότερου σταδίου. Φυσικά ο αριθμός των ασθενών που μελετήθηκαν δεν αρκεί για εξαγωγή συμπερασμάτων, αλλά τα παραπάνω αποτελέσματα συμφωνούν με τα γνωστά ως σήμερα ευρήματα για τον PAR1. Παραμένει να διευκρινιστεί η σημασία της αυξημένης έκφρασης του PAR1 σε ασθενείς με κακοήθεις όγκους στους πνεύμονες, αφού πρώτα επιβεβαιωθεί αυτή η αυξημένη έκφραση σε μεγαλύτερο αριθμό ασθενών.
From the onset of studies of PAR1, it has been concluded that this receptor is closely related to cancer. This relationship has been established after various experiments in cancer cell lines and in experimental animal models. The purposes of the present study can be summarized as follows: To explore the expression of PAR1 in cell lines established from human solid tumors and specifically PC3 and LNCaP from prostate cancer and MDA-231 and MCF-7 from breast cancer. To explore the suppression of PAR1 to the above cell lines in cell division. To determine if the activation of PAR1 to the above cell lines leads to MAPK phosphorylation. And ultimatilly, to explore the expression of PAR1 in patients that have been operated for tumor in lungs in Patras University Hospitall by Dr. D. Dougenis and colleagues. It was found that PAR1 is strongly expressed in highly metastatic cell lines PC3 and MDA-231, opposite to the cells LNCaP and MCF-7 that have lower metastatic capacity. The finding for the breast cancer cells MDA-231 and MCF-7 was according to published results (Kamath et al., 2001). PAR1 selective antagonist SCH 79797, reduced cell survival and DNA synthesis to all the above mentioned cell lines, independently of PAR1 expression. These non-specific results contributed to the recent fact that these antagonists were not PAR1 selective finally. Thrombin caused more than 100% induction of DNA synthesis in PC3 cells and had no effect in MDA-231 cells in accordance with published results that thrombin reduces the metastatic capacity of MDA-231 cells (Kamath et al., 2001). This effect of thrombin in PC3 cells, is mediated by activation of PAR1 as it was shown with the use of the selective agonist peptide SFLLRN. The activation of PAR1 by thrombin in PC3 cells leads to MAPK activation as it was shown by Western analysis. Furthrmore, preliminary experiments indicate that MAPK phosphorylation after PAR1 activation may be result of EGFR transactivation. In the sample tissues from patients, PAR1 expression was detected in all cancers with different ODs. The number of the samples is not enough to lead to conclusions, but there are some important observations. The highest level of PAR1 expression as was detected by RT-PCR were found to the sample tissues of the patient diagnosed for melanoma and of the patient with the most advanced stage of lung cancer. More patients shoulde be examined and more experiments to be done in order to proceed to conclusions for the significance of PAR1 in lung cancer.