Thimerosal is a mercury-containing component found in many vaccine preparations and used as a preservative. It causes Ca2+ movement across cell membrane in different cells that may be mediated via effects on different receptors and ion channels. A rise in intracellular free Ca2+ concentrations ([Ca2+]i) is a key signal for many pathophysiological processes in cells, including apoptosis and necrosis. Thimerosal increases [Ca2+]i in different cell types. The mechanisms underlying thimerosal-induced Ca2+ signal vary with cell types. The present study evaluated the effects of thimerosal on [Ca2+]i in human prostate cancer cells (PC3). WST-1 reduction assays and propidium iodide-staining assays were used to determine cell viability and apoptosis in the presence of thimerosal. The experimental results may be helpful to understand the pahrmocological and toxicological effects of thimerosal on cells from important organs. Results showed that thimerosal (10¡V200£gM) increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Thimerosal-induced Ca2+ influx was inhibited by econazole, SKF963656, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, a 200-mM thimerosal-induced [Ca2+]i rise was partly inhibited after pretreatment with 2,5-di-tert-butylhydroquinone (BHQ) (an endoplasmic reticulum Ca2+ pump inhibitor). Thimerosal at 1¡V7£gM induced cell death in a concentration-dependent manner that was not reversed when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N¡¦,N¡¦-tetraacetic acid (BAPTA). Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in PC3 cells, thimerosal induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Thimerosal also induced cell death in a Ca2+-independent apoptotic manner.

Depressive disorder is one of the most important diseases influencing human health in the 21st century. Antidepressants can improve some depressive symptoms and signs of depressive disorder in patients. It is thought that neurotransmitters (especially serotonin and/or norepinephrine and/or dopamine) have important roles in antidepression effects, but their pharmacological effects on the intracellular signal transduction pathway remain unclear. The aim of this thesis is to explore the effect of the antidepressants paroxetine (a selective serotonin reuptake inhibitor) and nortriptyline (a tricyclic antidepressant) on the intracellular Ca2+-related signal transduction and apoptosis of human prostate cancer PC3 cells. By using the fura-2 method, in PC3 cell, we found paroxetine (at concentrations between 10-150 £gM) and nortriptyline (at concentrations between 50-500 £gM) increased [Ca2+]i of PC3 cells in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor nearly abolished paroxetine and nortriptyline-induced Ca2+ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor-induced [Ca2+]i rise, suggesting that antidepressants released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C with U73122 inhibited paroxetine-induced [Ca2+]i rise by 80%, but did not change nortriptyline-induced [Ca2+]i rise. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. Nortriptyline-induced Ca2+ influx was inhibited by activation of protein kinase C. Nortriptyline at a concentration of 10 £gM increased the viability of the PC3 cells. At 50 £gM, nortriptyline killed 45% of the cells, and induced significant apoptosis, as measured by propidium iodide staining. Collectively, in PC3 cells, paroxetine induced [Ca2+]i rise is caused by phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Nortriptyline increased [Ca2+]i via phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx from the protein kinase C-sensitive pathway. Nortriptyline also induced apoptosis at a higher level. The results of this thesis may be helpful for understanding the effects of antidepressants on the intracellular signaling of cultured cells, and might illustrate a new possible mechanism concerning antidepressants¡¦ therapeutic effects or clinical side effects.

Nestas últimas décadas, investigações sobre compostos naturais, efetivos, não tóxicos, com potencial radioprotetor vêm despertando um grande interesse em consonância com a utilização crescente de vários tipos de radiação ionizante nas mais diversas finalidades. Entre eles a própolis uma resina coletada pelas abelhas (Apis mellifera), vem sendo apontada como promissora por apresentar uma série de características biológicas vantajosas, por exemplo, anti-inflamatória, antimicrobiana, antitumoral, imunomoduladora, antioxidante e também scavenger de radicais livres. O presente estudo teve como objetivo principal, averiguar efeito da própolis brasileira procedente de Rio Grande do Sul (AF 08) em células de ovário de hamster Chinês (CHO-K1) e em células tumorais de próstata (PC3), irradiadas com 60Co. Para tanto, foram utilizados três principais parâmetros interligados entre si: indução de micronúcleo, viabilidade celular e morte clonogênica. A escolha destes parâmetros se justifica pelo seu significado biológico, além do fato de serem prontamente observáveis e mensuráveis em células irradiadas. Os dados citogenéticos obtidos, mostraram um efeito radioprotetor da própolis (5 - 100 μg/ml) na indução de dano ao DNA em ambas as linhagens celulares, irradiadas com doses de 1 - 4 Gy. No entanto, o ensaio de citotoxicidade mostrou um efeito antiproliferativo pronunciado da própolis (50 - 400 μg/ml) em células PC3 irradiadas com 5 Gy. As curvas de sobrevida obtidas foram ajustadas satisfatoriamente pelo modelo linear-quadrático, cujo componente foi mais alto em células CHO-K1. Quanto à capacidade clonogênica, as células PC3 mostraram-se mais radiossensíveis que as células CHO-K1 nas doses mais altas da curva de sobrevida. A própolis, nas concentrações de 30 - 100 μg/ml, não influenciou no potencial clonogênico das células PC3, visto que, as curvas de sobrevida associadas ou não com a própolis, mostraram perfis similares, ao passo que o tratamento combinado em células CHO-K1 exibiu um efeito estimulador da proliferação. Os dados obtidos in vitro mostraram um potencial uso da própolis AF-08, como uma substância natural e não tóxica, na prevenção contra os efeitos danosos da radiação ionizante, nas doses e nas concentrações analisadas. / In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60Co radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400 μg/ml) in PC3 cells irradiated with 5 Gy. The survival curves obtained were adequately fitted by a linear-quadratic model, where the coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a potential use of propolis AF-08, a natural and nontoxic compound, in the prevention against the adverse effect of ionizing radiation, at the doses and concentrations here analyzed.

Nestas últimas décadas, investigações sobre compostos naturais, efetivos, não tóxicos, com potencial radioprotetor vêm despertando um grande interesse em consonância com a utilização crescente de vários tipos de radiação ionizante nas mais diversas finalidades. Entre eles a própolis uma resina coletada pelas abelhas (Apis mellifera), vem sendo apontada como promissora por apresentar uma série de características biológicas vantajosas, por exemplo, anti-inflamatória, antimicrobiana, antitumoral, imunomoduladora, antioxidante e também scavenger de radicais livres. O presente estudo teve como objetivo principal, averiguar efeito da própolis brasileira procedente de Rio Grande do Sul (AF 08) em células de ovário de hamster Chinês (CHO-K1) e em células tumorais de próstata (PC3), irradiadas com 60Co. Para tanto, foram utilizados três principais parâmetros interligados entre si: indução de micronúcleo, viabilidade celular e morte clonogênica. A escolha destes parâmetros se justifica pelo seu significado biológico, além do fato de serem prontamente observáveis e mensuráveis em células irradiadas. Os dados citogenéticos obtidos, mostraram um efeito radioprotetor da própolis (5 - 100 μg/ml) na indução de dano ao DNA em ambas as linhagens celulares, irradiadas com doses de 1 - 4 Gy. No entanto, o ensaio de citotoxicidade mostrou um efeito antiproliferativo pronunciado da própolis (50 - 400 μg/ml) em células PC3 irradiadas com 5 Gy. As curvas de sobrevida obtidas foram ajustadas satisfatoriamente pelo modelo linear-quadrático, cujo componente foi mais alto em células CHO-K1. Quanto à capacidade clonogênica, as células PC3 mostraram-se mais radiossensíveis que as células CHO-K1 nas doses mais altas da curva de sobrevida. A própolis, nas concentrações de 30 - 100 μg/ml, não influenciou no potencial clonogênico das células PC3, visto que, as curvas de sobrevida associadas ou não com a própolis, mostraram perfis similares, ao passo que o tratamento combinado em células CHO-K1 exibiu um efeito estimulador da proliferação. Os dados obtidos in vitro mostraram um potencial uso da própolis AF-08, como uma substância natural e não tóxica, na prevenção contra os efeitos danosos da radiação ionizante, nas doses e nas concentrações analisadas. / In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60Co radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400 μg/ml) in PC3 cells irradiated with 5 Gy. The survival curves obtained were adequately fitted by a linear-quadratic model, where the coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a potential use of propolis AF-08, a natural and nontoxic compound, in the prevention against the adverse effect of ionizing radiation, at the doses and concentrations here analyzed.

Η HARP είναι ένας αυξητικός παράγοντας με πλειοτροπική δράση που εμπλέκεται στην ανάπτυξη, τη διαφοροποίηση και τη μετανάστευση πολλών ειδών κυττάρων καθώς και στην αγγειογένεση και την καρκινογένεση. Η HARP ασκεί τις δράσεις της μέσω των διαμεμβρανικών της υποδοχέων RPTPβ/ζ, ALK και Ν-συνδεκάνη. Η δομή της HARP αποτελείται από δύο κεντρικές περιοχές ομόλογες με τις επαναλαμβανόμενες περιοχές τύπου I της θρομβοσπονδίνης (Thrombospondin type I Repeat, TSR) και το αμινοτελικό και καρβοξυτελικό της άκρο που έχουν τυχαία διαμόρφωση στο χώρο. Έχει βρεθεί ότι η HARP αποτελεί υπόστρωμα διάφορων πρωτεολυτικών ενζύμων και πεπτίδια της έχουν ανιχνευθεί στο υγρό θρεπτικό μέσο σε καλλιέργειες διαφόρων κυττάρων. Τα πεπτίδια της HARP μπορούν να έχουν διαφορετική ή και αντίθετη δράση από αυτή ολόκληρου του μορίου. Στo πλαίσιo της μελέτης των επιμέρους περιοχών της HARP που συμμετέχουν στις βιολογικές δράσεις της αλλά και της δράσης των πεπτιδίων που προκύπτουν από την πρωτεόλυσή της, σε αυτή την εργασία μελετήθηκε η δράση δύο ανασυνδυασμένων, τροποποιημένων μορφών της HARP που αντιστοιχούν στις κεντρικές TSR περιοχές, των H9-59 και H60-110, στον πολλαπλασιασμό και την προσκόλληση κυττάρων καρκίνου του προστάτη PC3 και στη φωσφορυλίωση των μορίων μεταγωγής σήματος ERK1/2. Τα αποτελέσματα μας έδειξαν ότι τα πολυπεπτίδια H9-59 και H60-110 καταστέλλουν τον πολλαπλασιασμό των κυττάρων PC3, ενώ επάγουν την προσκόλληση των ίδιων κυττάρων. Επιπλέον, τόσο το H9-59 όσο και το H60-110 επάγουν τη φωσφορυλίωση των ERK1/2. / HARP is a heparin-binding growth factor, which plays a key role in cell proliferation, differentiation and migration. HARP is also involved in angiogenesis and tumor growth and progression. HARP mediates its functions through its transmembrane receptors RPTPβ/ζ, ALK and N-syndecan. Structurally, HARP contains two TSR homologous domains and two basic clusters in its N and C-termini. It has been found that HARP is a substrate for several extracellular proteolytic enzymes and HARP peptides have been detected in conditioned media of cells. HARP peptides often have different or even opposite biological activity from the whole molecule. The aim of this project was to study the biological activity of two recombinant, truncated forms of HARP that correspond to the two TSR domains, H9-59 and H60-110, in order to contribute to the study of the HARP domains that mediate its biological activities and also the biological activity of HARP peptides. The results of the study show that both H9-59 and H60-110 inhibit the proliferation of PC3 cells but stimulate their adhesion. Moreover, both H9-59 and H60-110 stimulate the phosphorylation of ERK1/2.

Our recent studies have shown that that erectile dysfunction (ED) drugs including Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) enhance killing of several types of cancer cells by anticancer drug, Doxorubicin (DOX). We observed increased cell death by apoptosis in response to the combined treatment with ED drugs and DOX. However, the mechanism of such enhancement of cell death by combined treatment of ED drugs and DOX is not fully understood. Nuclear factor-κB (NF-κB) is an oxidant-sensitive transcription factor that plays a critical role in the immediate-early activation of a multitude of genes that have been documented to play critical role in programmed cell death (apoptosis). NF-κB activation has been shown to block apoptosis and its inhibition improves existing anti-oncogenic therapy such as chemotherapy. In the present study, we tested the hypothesis whether combined treatment of prostate cancer cells, PC3, with Sildenafil plus DOX would attenuate the activation of NFκB by inhibiting translocation of the p65 and p50 subunits to the nucleus and by phosphorylation of cytosolic IκB In addition, we investigated the effect of DOX and DOX plus Sildenafil on the expression of BCL family of proteins which play critical role in apoptosis. We treated PC3 cells with 1.5 μM DOX with or without 10 µM Sildenafil for 6 hours and 72 hours. The nuclear translocation of p65 and p50 and expression of BCL family of proteins was determined by western blot analysis. Our results show that combined treatment of DOX and Sildenafil significantly reduced the nuclear translocation of p65 and p50 as compared with DOX alone (P < 0.05). This correlated with the significant reduction in the expression of Bcl-2, BclxL and phosphorylation of BAD. These data provide an important mechanism by which Sildenafil treatment augments the apoptotic potential of DOX in PC3 cancer cells.

Die Cathepsine B, H und L sind lysosomale Enzyme, die zur Gruppe der Cysteinproteasen gehören. Erhöhte Aktivitäten dieser Proteasen fand man in Gewebeproben verschiedener Tumore, vermutlich Hinweis auf eine Beteiligung an Invasion und Metastasierung. Unter physiologischen Bedingungen werden die Cathepsine von endogenen Inhibitoren kontrolliert, eine Verminderung dieser Cysteinprotease-Inhibitoren (CPI) würde die proteolytische Dysbalance verstärken und zur Ausbreitung des malignen Prozesses beitragen. Für das Prostatakarzinom gab es bisher keine Untersuchungen. Die Aktivität der Cathepsine wurde mit Hilfe spezifischer Substrate bestimmt, die zur Bildung fluorogener Produkte führten. Zur Bestimmung der inhibitorischen Aktivität der CPI wurden die Proben nach einer Hitzeaktivierung gegen reines Cathepsin B getestet. Untersucht wurden Gewebeproben verschiedener Patientengruppen, Primärzellkulturen, die aus normalem und maligne veränderten Prostatagewebe angezüchtet wurden und vergleichend dazu die drei immortalisierten Zellinien LNCaP, PC3 und DU145. Die Ergebnisse der Gewebeproben zeigten höhere Aktivitäten der Cathepsine B und L im nichterkrankten Gewebe, nicht wie erwartet im Tumorgewebe der Prostata. Hingegen waren bei den Primärzellkulturen alle drei Cathepsine und der Quotient Cathepsine/CPI in den Tumorproben erhöht. Die immortalisierten Zellinien zeigten die gleiche Verteilung bei allen Cathepsinen, DU145 mit der höchsten Aktivität, gefolgt von LNCaP und PC3. Anhand der Ergebnisse schlußfolgern wir, daß die Untersuchung von Gewebe-proben der Prostata hinsichtlich der Beteiligung der Cathepsine am Tumor-geschehen keine eindeutigen Erkenntnisse erbringt. Dies ist vermutlich auf die Heterogenität des Gewebes zurückzuführen, das nicht nur epitheliale, sondern auch stromale Zellen enthält. Die aus dem Gewebe angezüchteten Primär-zellkulturen scheinen ein genaueres Bild des Verhältnisses von Cathepsinen und den Inhibitoren zu geben und sind unserer Meinung nach den Bestimmungen in Gewebeproben vorzuziehen. / The cathepsins B, H and L (CB, CH, CL) are lysosomal proteolytic enzymes belonging to the cysteine protease family. Elevated cathepsin levels and decreased concentration of their endogenous inhibitors have been demonstrated in a variety of tumors, suggesting a contribution to invasion and metastasis. The situation for prostate cancer was so far unknown. Using fluorimetric assays, catalytic activities of the cathepsins B, H, L were measured in prostatic tissue samples obtained from different groups of patients, in primary cell cultures established from human prostate and in the immortalized cell lines LNCaP, PC3 and DU145. Inhibitory activities of cysteine protease inhibitors (CPI) were tested against purified cathepsin B. Comparing matched pairs of normal and cancerous tissue samples from the prostate, significantly decreased levels of CB and CL were found in malignant samples. In contrast, primary cell cultures from malignant tissue showed elevated levels of all three cathepsins and increased ratios of cathepsins to CPI when compared to cell cultures from non-malignant prostate. The permanent cell lines showed a similiar distribution of cathepsin levels, DU145 with the highest activity, followed by LNCaP and PC3. These results suggest that elevated cathepsin activities and increased ratios of cathepsins to CPI in malignant cell cultures compared to non-malignant samples may be an indication for a cellular proteolytic imbalance in prostatic cancer cells. Regarding different results, determinations in primary cell cultures should be preferred to tissue samples.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata. / It has been reported that accumulation of mutation on proto-oncogenes and tumor suppressor genes directs cells to carcinogenesis. In most described cancer, cells display uncontrolled proliferation due to altered expression or mutations of cyclins, cyclin-dependent kinases and cell cycle inhibitors. The solid tumors are the most common cancer type in world. Chemotherapy and/or hormone-therapy, radiotherapy and surgery are the suitable treatment for this disease. However, chemotherapy has been shown several side effects and often ineffective. Therefore, the search for new molecules with antitumoral activity low cytotoxicity is needed. The aim of this work was to evaluate the in vitro antitumoral effects of a new synthetic compound, the pterocarpanquinone LQB 118, on tumor cell lines of high prevalence in the world and to study some mechanisms of action. Tumor cell lines of breast (MCF7), lung (A549) and prostate (PC3) were cultivated at RPMI medium with 10% of serum fetal bovine. The cytotoxicity was evaluated by MTT assay and the cell proliferation by cell counting (trypan blue exclusion) and cell cycle analysis (flow cytometry). Apoptosis was evaluated by chromatin condensation (fluorescence microscopy with DAPI), DNA fragmentation (electrophoresis) and annexin-V and iodide propidium staining. Gene expression was studied by RT-PCR. As LQB 118 (5 g/ml) induced cytotoxic effect mainly on prostate tumor cells, further experiments were then performed only with this tumor cell line. The LQB 118 cytotoxic effects were time and concentration-dependent. Furthermore, this substance inhibited cell proliferation and promoted cell cycle arrest, increasing cell number in S and G2/M phases. Studying the mechanisms of the LQB 118 antitumoral action, it was demonstrated that this substance inhibited the mRNA expression of the transcription factor c-Myc, cyclin D1 and cyclin B1 and also induced apoptosis of PC3. Concluding, LQB 118 impairs prostate tumor cells proliferation due to altered mRNA expression of some cell cycle regulator genes, resulting in cell cycle arrest and apoptosis induction, suggesting this compound as a good candidate for a future drug in prostate cancer treatment.

Dissertationsschrift zur Erlangung eines doctor medicinae (Dr.med.) der Medizinischen Fakultät Carl Gustav Carus der Technischen Universität Dresden

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata. / It has been reported that accumulation of mutation on proto-oncogenes and tumor suppressor genes directs cells to carcinogenesis. In most described cancer, cells display uncontrolled proliferation due to altered expression or mutations of cyclins, cyclin-dependent kinases and cell cycle inhibitors. The solid tumors are the most common cancer type in world. Chemotherapy and/or hormone-therapy, radiotherapy and surgery are the suitable treatment for this disease. However, chemotherapy has been shown several side effects and often ineffective. Therefore, the search for new molecules with antitumoral activity low cytotoxicity is needed. The aim of this work was to evaluate the in vitro antitumoral effects of a new synthetic compound, the pterocarpanquinone LQB 118, on tumor cell lines of high prevalence in the world and to study some mechanisms of action. Tumor cell lines of breast (MCF7), lung (A549) and prostate (PC3) were cultivated at RPMI medium with 10% of serum fetal bovine. The cytotoxicity was evaluated by MTT assay and the cell proliferation by cell counting (trypan blue exclusion) and cell cycle analysis (flow cytometry). Apoptosis was evaluated by chromatin condensation (fluorescence microscopy with DAPI), DNA fragmentation (electrophoresis) and annexin-V and iodide propidium staining. Gene expression was studied by RT-PCR. As LQB 118 (5 g/ml) induced cytotoxic effect mainly on prostate tumor cells, further experiments were then performed only with this tumor cell line. The LQB 118 cytotoxic effects were time and concentration-dependent. Furthermore, this substance inhibited cell proliferation and promoted cell cycle arrest, increasing cell number in S and G2/M phases. Studying the mechanisms of the LQB 118 antitumoral action, it was demonstrated that this substance inhibited the mRNA expression of the transcription factor c-Myc, cyclin D1 and cyclin B1 and also induced apoptosis of PC3. Concluding, LQB 118 impairs prostate tumor cells proliferation due to altered mRNA expression of some cell cycle regulator genes, resulting in cell cycle arrest and apoptosis induction, suggesting this compound as a good candidate for a future drug in prostate cancer treatment.