<p>Colorectal cancer is one of the most common malignant diseases in Sweden – more than 5000 new cases are diagnosed each year. The overall five-year survival is about 60% and in cases of recurrence the prognosis is poor.</p><p>In a phase III study in advanced colorectal cancer the response rate was doubled when 5-FU was given as a bolus injection versus as a short infusion. The toxicity was similar and time to progression was longer in the injection group. However, overall survival was not significantly different. Dose-effect relationships of 5-FU were studied in another phase III study recruiting 312 patients. A decrease from 500 mg/m² to 400 mg/m² worsened the treatment results. A low incidence of severe toxicity was seen in both groups. An increase to 600 mg/m² worsened the toxicity without any improvement of the results.</p><p>A cytotoxic drug sensitivity test in different tumour types, mainly gastrointestinal cancer, poorly predicted treatment outcome in a phase II study.</p><p>The conventional Nordic Flv regimen was split in a phase I/II trial. An escalation of dose was possible and the response rate was 20%.</p><p>Thymidylate synthase (TS) and the gene expression of p53 were investigated by immunohistochemical technique in the primary tumours of 132 patients. None of the markers predicted the later palliative chemotherapy result. However, TS significantly predicted time to recurrence.</p><p>Serum markers were analysed before and during FLv treatment to early predict outcomes among 87 patients. TPS is promising, both as a predictive marker before start of treatment and after a short period of treatment. In the same setting, CEA had lower predictive value. S-VEGF and S-bFGF did not yield any prognostic information of later outcome. In all studies B-haemoglobin values, performance status and subjective response were strong markers, both for prediction of objective response and for survival.</p>

Colorectal cancer is one of the most common malignant diseases in Sweden – more than 5000 new cases are diagnosed each year. The overall five-year survival is about 60% and in cases of recurrence the prognosis is poor. In a phase III study in advanced colorectal cancer the response rate was doubled when 5-FU was given as a bolus injection versus as a short infusion. The toxicity was similar and time to progression was longer in the injection group. However, overall survival was not significantly different. Dose-effect relationships of 5-FU were studied in another phase III study recruiting 312 patients. A decrease from 500 mg/m2 to 400 mg/m2 worsened the treatment results. A low incidence of severe toxicity was seen in both groups. An increase to 600 mg/m2 worsened the toxicity without any improvement of the results. A cytotoxic drug sensitivity test in different tumour types, mainly gastrointestinal cancer, poorly predicted treatment outcome in a phase II study. The conventional Nordic Flv regimen was split in a phase I/II trial. An escalation of dose was possible and the response rate was 20%. Thymidylate synthase (TS) and the gene expression of p53 were investigated by immunohistochemical technique in the primary tumours of 132 patients. None of the markers predicted the later palliative chemotherapy result. However, TS significantly predicted time to recurrence. Serum markers were analysed before and during FLv treatment to early predict outcomes among 87 patients. TPS is promising, both as a predictive marker before start of treatment and after a short period of treatment. In the same setting, CEA had lower predictive value. S-VEGF and S-bFGF did not yield any prognostic information of later outcome. In all studies B-haemoglobin values, performance status and subjective response were strong markers, both for prediction of objective response and for survival.

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Made available in DSpace on 2016-08-17T18:39:34Z (GMT). No. of bitstreams: 1 3220.pdf: 7184046 bytes, checksum: d31437c1aa1937336c7b8cb91918b19b (MD5) Previous issue date: 2010-08-06 / Universidade Federal de Minas Gerais / Malaria disease, caused mainly by Plasmodium falciparum parasite, afflicts about 500 million people and causes nearly one million deaths every year. For the development of new drugs against this disease, one possible approach is to identify an enzyme that plays a key role in P. falciparum development and presents significantly different properties from the corresponding human one. These differences can be exploited in the design of specific inhibitors of the parasite s protein, thus, three different enzymes were selected as possible targets. As there are evidences suggesting that increasing oxidative stress can effectively inhibit the growth of the malarial parasite the enzyme Glutathione Reductase of P. falciparum (PfGR), responsible for the parasite s antioxidant defense, has become a potential target for the design and development of inhibitors. The second target was the P. falciparum Dihydrofolate Reductase-Thymidylate Synthase (PfDHFR-TS), and in this case blocking its action stops the dTMP production and DNA synthesis in the parasite. The third chosen target was the P. falciparum Lactate Dehydrogenase (PfLDH), whose inhibition interrupts the ATP formation and thus causing the death of the parasite. So that a family of arilloxazines compounds, together with chloroquine and methylene blue, were studied by means of docking simulations in the binding sites of these enzymes and also in the corresponding human enzymes for comparison. The three-dimensional structures of the enzymes and of chloroquine and methylene blue were obtained from the Protein Data Bank (PDB). The structures of the arilloxazines compounds, in turn, were obtained by molecular modeling with HyperChem 6.01 and MOPAC2009 programs, using as starting models similar crystallographic structures deposited in the Cambridge Structural Database. Docking simulations were performed using GOLD 4.0.1. The docking results showed that the enzymes PfGR and PfDHFR-TS are not the preferential targets of chloroquine. For the methylene blue it was possible to elucidate its binding mode in hGR and PfGR. Regarding the arilloxazines it was possible to show that they present their higher affinity for hGR, followed by PfGR, hDHFR, PfDHFR-TS, PfLDH and hLDH. In the case of GRs, the interface site was the preferred binding site. The results suggest that if arilloxazines compounds with higher affinity for PfGR are desirable then a pentafluorophenyl should be attached at the N10 position, as in the 2e compound. When searching for arilloxazines with higher affinity for PfLDH, it seems to be desirable a carboxymethyl group at the N3 position (as in 5b) and a pentafluorophenyl group at N10 (as in 2e). Finally, the results suggest that in general the studied arilloxazines probably will present a higher affinity for hDHFR than PfDHFR-TS. All these results are an important starting point for the design of new arilloxazines ligands so that they can be used as lead compounds in the search for new drugs against malaria. / A malária, causada principalmente pelo Plasmodium falciparum, atinge cerca de 500 milhões de pessoas e causa aproximadamente um milhão de mortes todos os anos. Para o desenvolvimento de novos fármacos contra esta doença, uma das abordagens possível é identificar uma enzima que desempenhe papel vital no desenvolvimento do P. falciparum e apresente propriedades significantemente diferentes das enzimas humanas correspondentes, de modo que tais diferenças possam ser exploradas no design de inibidores específicos à proteína do parasita. Existem evidências sugerindo que aumentar o estresse oxidativo pode inibir eficientemente o crescimento do parasita causador da malária e, portanto, a enzima Glutationa Redutase do P. falciparum (GRPf), responsável por sua defesa antioxidante, tornou-se um alvo em potencial para o desenvolvimento de inibidores. Também, o bloqueio da ação da Diidrofolato Redutase-Timidilato Sintase do P. falciparum (DHFR-TSPf) interrompe a produção de dTMP e a síntese de DNA no parasita. Ainda, espera-se que a inibição da Lactato Desidrogenase do P. falciparum (LDHPf) interrompa a produção de ATP no parasita e, consequentemente, cause sua morte. Portanto, estudou-se o comportamento de compostos da família das ariloxazinas, da cloroquina e do azul de metileno nos sítios de ligação destas enzimas, além das enzimas humanas correspondentes para fins de comparação, por meio de cálculos de docking. As estruturas tridimensionais das enzimas foram obtidas no Protein Data Bank (PDB). As estruturas dos inibidores da família das ariloxazinas, por sua vez, foram obtidas por meio de modelagem molecular, utilizando os programas HyperChem 6.01 e MOPAC2009, a partir de estruturas cristalográficas semelhantes obtidas no Cambridge Structural Database; já as estruturas da cloroquina e do azul de metileno foram obtidas também no PDB. Os cálculos de docking destes compostos nos sítios de ligação das enzimas estudadas foram realizados utilizando o programa GOLD 4.0.1. Com base nos resultados de docking, sugere-se que as enzimas GRPf e DHFR-TSPf não são alvos preferenciais da cloroquina. Também, pôde-se elucidar o possível modo de ligação do azul de metileno nas enzimas GRh e GRPf. No geral, foi possível sugerir ainda que as ariloxazinas devam apresentar maior afinidade pela GRh, seguida por GRPf, DHFRh, DHFR-TSPf, LDHPf e LDHh, nesta ordem. Nas GRs, o sítio da interface foi o sítio preferencial de ligação. Para se buscar inibidores da família das ariloxazinas com maior afinidade pela GRPf, sugere-se considerar um pentafluorfenil como substituinte na posição N10, como no composto 2e. Ainda, na busca por ariloxazinas com maior afinidade pela LDHPf, sugere-se considerar um carboximetil na posição N3 (como o de 5b) e um pentafluorfenil na posição N10 (como em 2e). Por fim, foi obtido que as ariloxazinas estudadas possivelmente apresentarão, em geral, uma maior afinidade pela DHFRh do que pela DHFR-TSPf. Estes dados podem ser tomados como ponto de partida para o design de novos compostos da família das ariloxazinas, a fim de que possam atuar como compostos líderes na busca por novos fármacos contra a malária.

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Acquired cellular resistance to traditional chemotherapeutics is a common obstacle in the treatment of most cancer cell types. This resistance occurs as a result of changes in the underlying molecular mechanisms of disease progression. The development of novel chemotherapeutic approaches designed to enhance the efficacy of protypical anti-cancer drugs is important in order to overcome this issue. Such approaches will aid in understanding the biomolecular phenomena responsible for drug resistance and disease progression. Combining signaling pathway inhibitors has become an effective strategy for enhancing tumor cell death by targeting multiple pathways known to regulate cell survival. Pemetrexed, an FDA-approved anti-folate drug, targets thymidylate synthase (TS) and a secondary folate-dependent enzyme, 5’ aminoimidazole-carboximide ribonucleotide formyltransferase (AICART); both important for DNA synthesis. Studies performed by our collaborator demonstrated that TS inhibition causes intracellular accumulation of ZMP+ and activation of AMPK which is known to induce autophagy in mammalian cells. Previous studies from our lab and others showed that sorafenib, a multi-kinase inhibitor of Raf-1 and class III receptor tyrosine kinases, was able to induce a cytotoxic form of autophagy in a variety of tumor cell types. Combination treatment using pemetrexed and sorafenib in these cancer cells resulted in an enhancement of autophagy and cell lethality beyond that of individual drugs alone. Inhibition of autophagy suppressed the toxic interactions of these drugs in all cell types examined. Pemetrexed/sorafenib cotherapy also proved to be an effective treatment for triple negative breast cancer cells having advanced to a stage of estrogen independence. Fulvestrant-resistant MCF7 cells were more sensitive to the drug combination than parental, estrogen-dependent MCF7 cells. Breast cancer cells cotreated with pemetrexed and sorafenib exhibited enhanced MEK/ERK signaling, Src activation that was dependent on platelet-derived growth factor β (PDGFRβ) downregulation, elevated protein phosphatase 2A (PP2A) activity, and increased de novo ceramide synthesis. Studies using a mouse model of experimentally-induced breast cancer validated drug combination effectiveness through inhibition of tumor growth, while no deleterious effects on normal tissues were observed. The data presented demonstrates that pemetrexed/sorafenib cotreatment augments chemosensitivity in both in vitro and in vivo systems. Based upon these findings, a Phase I clinical trial involving pemetrexed and sorafenib in breast cancer patients with solid, recurrent tumors was begun in 2011. In conclusion, this work strongly supports a promising therapeutic utility for the pemetrexed/sorafenib combination in treatment of various cancer cell types.

O objetivo do estudo foi estudar a expressão de timidilato sintase (TS) como fator preditivo para eficácia de quimioterapia adjuvante com 5-fluorouracil (5-FU) e como fator prognóstico para sobrevida em pacientes portadores de câncer colorretal. Trata-se de estudo retrospectivo em uma série de 114 pacientes com carcinoma colorretal estádios II ou III, distribuídos em dois grupos: 1)cirurgia exclusiva (n=61); 2)cirurgia seguida de quimioterapia com 5-FU (n=53). A expressão de TS foi determinada por imunoistoquímica. Observou-se que a expressão intratumoral de TS foi capaz de selecionar pacientes que se beneficiaram com emprego de quimioterapia adjuvante, mas não se mostrou como variável independente para risco de recidiva ou óbito / The purpose of this study was trying to assess the value of TS expression as a predictive factor in the efficacy of adjuvant chemotherapy in colorectal cancer, as well as its independent prognostic value for survival. It deals with a retrospective study that assesses a series of 114 individuals with high risk colorectal cancer, distributed into two different groups: 1)surgery alone (n=61); 2)surgery and 5-FU-based chemotherapy (n=53). TS expression was determined by immunohistochemistry. We observed that TS expression may select patients that benefit from adjuvant chemotherapy, but it was not shown as an independent variable for the risk of recurrence or death

O objetivo do estudo foi estudar a expressão de timidilato sintase (TS) como fator preditivo para eficácia de quimioterapia adjuvante com 5-fluorouracil (5-FU) e como fator prognóstico para sobrevida em pacientes portadores de câncer colorretal. Trata-se de estudo retrospectivo em uma série de 114 pacientes com carcinoma colorretal estádios II ou III, distribuídos em dois grupos: 1)cirurgia exclusiva (n=61); 2)cirurgia seguida de quimioterapia com 5-FU (n=53). A expressão de TS foi determinada por imunoistoquímica. Observou-se que a expressão intratumoral de TS foi capaz de selecionar pacientes que se beneficiaram com emprego de quimioterapia adjuvante, mas não se mostrou como variável independente para risco de recidiva ou óbito / The purpose of this study was trying to assess the value of TS expression as a predictive factor in the efficacy of adjuvant chemotherapy in colorectal cancer, as well as its independent prognostic value for survival. It deals with a retrospective study that assesses a series of 114 individuals with high risk colorectal cancer, distributed into two different groups: 1)surgery alone (n=61); 2)surgery and 5-FU-based chemotherapy (n=53). TS expression was determined by immunohistochemistry. We observed that TS expression may select patients that benefit from adjuvant chemotherapy, but it was not shown as an independent variable for the risk of recurrence or death

La fonction d'une protéine est souvent subordonnée à l'interaction avec un certain nombre de partenaires. L'étude de la structure tridimensionnelle de ces complexes, qui ne peut souvent se faire expérimentalement, permettrait la compréhension de nombreux processus cellulaires. Le travail présenté ici se compose de deux parties. La première traite de la mise en place d'une fonction de score pour l'amarrage protéine-protéine et la deuxième de l'étude cristallographique d'une protéine tétramérique qui est une cible antibiotique potentielle : la thymidylate synthase X de Paramecium bursaria Chlorella virus. La modélisation des complexes protéine-protéine ou docking comporte deux étapes successives : d'abord, un grand nombre de conformations sont générées, puis une fonction de score est utilisée pour les classer. Cette fonction de score doit prendre en compte à la fois la complémentarité géométrique des deux molécules et les propriétés physico-chimiques des surfaces en interaction. Nous nous sommes intéressés à la seconde étape à travers le développement d'une fonction de score rapide et fiable. Ceci est possible grâce à la tessellation de Voronoï de la structure tridimensionnelle des protéines. En effet, les tessellations de Voronoï ou de Laguerre se sont avérées être de bons modèles mathématiques de la structure des protéines. En particulier, cette formalisation permet de faire une bonne description de l'empilement et des propriétés structurales des résidus. Cette modélisation rend compte l'empilement des résidus à l'interface entre deux protéines. Ainsi, il est possible de mesurer un ensemble de paramètres sur des complexes protéine-protéine dont la structure est connue expérimentalement et sur des complexes leurres générés artificiel- lement. Ces paramètres, sont la fréquence d'apparition des résidus ou des paires de résidus, les volumes des cellules de Voronoï, les distances entre les résidus en contact à l'interface, la surface de l'interface et le nombre de résidus à l'interface. Ils ont été utilisés en entrée de procédures d'apprentissage statistique. Grâce à ces procédures (apprentissage logistique, séparateurs à vaste marge (SVM) et algorithmes génétiques), on peut obtenir des fonctions de score efficaces, ca- pables de séparer les leurres des structures réelles. Dans un deuxième temps, j'ai déterminé expérimentalement la structure de la thymidylate synthase X, cible antibiotique de choix. La thymidylate synthase X est une flavoprotéine qui a été découverte récemment. Elle intervient dans la synthèse du dTMP chez la plupart des procaryotes mais n'existe pas chez les eucaryotes supérieurs. Cette protéine catalyse le transfert de methyle du tétrahydrofolate vers le dUMP grâce à son cofacteur le FAD et au NADPH qui intervient comme substrat. La structure tridimensionnelle de l'homotétramère de la thymidylate synthase X en présence de son cofacteur, le FAD, a été résolue à 2.4 Å par remplacement moléculaire. Comme pour les structures de thymidylate synthase X de Thermotoga maritima et de Mycobacterium tuberculosis précédemment résolues, le monomère se compose d'un coeur de feuillets β et de deux hélices α à son extrémité. Le site actif se trouve à l'interface de trois monomères, la partie isoalloxazine du FAD étant accessible au solvant et proche d'une longue boucle flexible. La fixation du FAD dans cette structure est légèrement différente de celles déjà observées par la conformation de la partie adénine. Cette structure, associée aux études de mutagénèse dirigée de nos collaborateurs, a permis de mettre évidence des résidus jouant un rôle majeur lors de la catalyse.