Assessment of Notch1 ligands production - key protein targets in breast cancer

Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer. To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands. We aim to determine the best expression and purification conditions for the different ligand protein constructs, to develop specific function-blocking antibodies using the Phage Display technology. Moreover, we propose to crystallize the Notch1 ligands alone and in complex with the phage display selected antibodies, unveiling molecular details. hJag2DE3 and hDll1DE6 proteins were purified from refolded inclusion bodies or mammalian cell culture supernatants, respectively, and purity was confirmed by SDS-PAGE (>95%). Protein produced in mammalian cells showed to be more stable, apparently with the physiological disulfide pattern, contrary to what was observed in the refolded protein. Several nano-scale crystallization experiments were set up in 96-well plates, but no positive result was obtained. We will continue to pursue for the best expression for the Notch ligand constructs in both expression systems.

Phage display as a tool for development of novel therapeutics for breast cancer

Phage display technology is a powerful platform for the generation of highly specific human monoclonal antibodies (Abs) with potential use in clinical applications. Moreover, this technique has also proven to be a reliable approach in identifying and validating new cancer-related targets. For scientific or medical applications, different types of Ab libraries can be constructed. The use of Fab Immune libraries allows the production of high quality and affinity antigen-specific Abs. In this work, two immune human phage display IgG Fab libraries were generated from the Ab repertoire of 16 breast cancer patients, in order to obtain a tool for the development of new therapeutic Abs for breast cancer, a condition that has great impact worldwide. The generated libraries are estimated to contain more than 108 independent clones and a diversity over 90%. Libraries validation was pursued by selection against BSA, a foreign and highly immunogenic protein, and HER2, a well established cancer target. Preliminary results suggested that phage pools with affinity for these antigens were selected and enriched. Individual clones were isolated, however, it was not possible to obtain enough data to further characterize them. Selection against the DLL1 protein was also performed, once it is a known ligand of the Notch pathway, whose deregulation is associated to breast cancer, making it an interesting target for the generation of function-blocking Abs. Selection resulted in the isolation of a clone with low affinity and Fab expression levels. The validation process was not completed and further effort will have to be put in this task in the future. Although immune libraries concept implies limited applicability, the library reported here has a wide range of use possibilities, since it was not restrained to a single antigen but instead thought to be used against any breast cancer associated target, thus being a valuable tool.

Thomsen-Friedenreich antigens in bladder cancer: evaluation of their prognostic value

RESUMO: Introdução. O cancro de bexiga é uma patologia comum que representa o 6° e o 5° cancro mais incidente em Portugal e na Itália, respetivamente. Em mais de metade dos casos ocorre reincidência durante o primeiro ano, requerendo acompanhamento clínico ao longo da vida. A instilação intravesical de Bacillus Calmette-Guérin (BCG) (uma estirpe atenuada do Mycobacterium bovis) representa uma imunoterapia eficaz no combate ao cancro de bexiga, no entanto, muitos aspetos da interação de BCG com as células tumorais bem como com as células do sistema imunitário permanecem por desvendar. As células tumorais de bexiga expressam frequentemente as formas sialiladas dos antigénios de Thomsen-Friedenreich (TF), i.e., sialil-T (sT) e sialil-Tn (sTn). Contudo ainda se desconhece o significado da sua expressão na malignidade tumoral e se afeta a eficácia da terapêutica BCG. Objetivo do estudo. Investigar o papel dos antigénios sT e sTn no fenótipo maligno de células de cancro de bexiga bem como na resposta mediada pelo sistema imunitário à terapia com BCG. Metodologia. Para tal, foram utilizadas as linhas celulares de cancro da bexiga HT1376 e MCR, geneticamente modificadas por transdução com vetores codificantes para as sialiltransferases ST3GAL1 ou ST6GALNAC1, de forma a expressar homogeneamente os antigénios sT ou sTn respetivamente. Estes modelos celulares foram estudados após confronto com BCG. O nível de BCG internalizado foi avaliado por citometria de fluxo. O perfil global de expressão genética dos modelos celulares antes e após incubação com BCG foi analisado pela tecnologia de microarray. O perfil de citocinas secretadas pelos modelos celulares após incubação com BCG, bem como de macrófagos estimulados pelo secretoma de células de cancro de bexiga que por sua vez foram estimuladas previamente por BCG, foi estudado pelo sistema multiplex de “imuno-esferas”. Resultados. A análise do transcritoma dos modelos celulares revelou que grupos de genes envolvidos em funções específicas foram modulados em paralelo nos dois modelos celulares, após transdução, independentemente da sialiltransferase expressa. Ou seja, em células que expressavam a sialiltransferase ST3GAL1 ou ST6GALNAC1, os genes envolvidos na regulação da segregação cromossómica e na reparação do DNA foram consistentemente regulados negativamente. Genes descritos na literatura como marcadores para o cancro de bexiga foram também modulados. A incubação com BCG resultou numa tendência ao aumento da expressão de genes relevantes na preservação e estabilidade genómica e menor malignidade, no entanto, apenas em células que expressavam sT ou sTn. Entre as dez citocinas testadas, apenas a IL-6 e IL-8 foram expressas pelas linhas celulares de cancro da bexiga, com indução destas após estimulação com BCG, e principalmente em células que expressavam ST3GAL1 ou ST6GALNAC1. Em macrófagos, citocinas inflamatórias, tais como IL-1β, IL-6 e TNFα, e a citocina anti-inflamatória IL-10, foram induzidas apenas pelo secretoma de células de cancro da bexiga confrontadas com BCG, com maior relevância quando estas expressavam ST3GAL1 ou ST6GALNAC1, prevendo a estimulação de macrófagos semelhantes aos de tipo M1 e uma melhor resposta à terapia com BCG. Conclusões. O efeito geral da expressão destas sialiltransferases e dos produtos enzimáticos sT ou sTn nas células de cancro de bexiga conduz a um fenótipo de maior malignidade. Contudo, a maior avidez de estas na produção de citocinas inflamatórias após confronto com BCG, bem como a maior capacidade de estimulação de macrófagos, predirá uma resposta à terapia com BCG mais eficaz em tumores que expressem os antigénios de TF sialilados. Tais conclusões são totalmente concordantes com os nossos mais recentes dados clínicos obtidos em colaboração, que mostram que em doentes com cancro de bexiga que expressam sTn respondem melhor a terapia BCG. ----------ABSTRACT: Background. Bladder cancer is a common malignancy representing the 6th and the 5th most incident cancer in Portugal and in Italy, respectively. More than half of the cases relapse within one year, requiring though a lifelong follow-up. Intravesical instillation of Bacillus Calmette-Guérin (BCG) (an attenuated strain of Mycobacterium bovis) represents an effective immunotherapy of bladder cancer, although many aspects of the interaction of BCG with cancer cells and host immune cells remain obscure. Bladder cancer cells often express the sialylated forms of the Thomsen-Friedenreich (TF), i.e., sialil-T (sT) e sialil-Tn (sTn). However, it’s still unknown the sense of such expression in tumour malignancy and in the BCG therapy efficacy. Aim of the study. To investigate the role of the sT and sTn antigens on the malignant phenotype of bladder cancer cells and the immune mediated response to BCG therapy. Experimental. We have utilized populations of the bladder cancer cell lines HT1376 and MCR, genetically modified by transduction with the sialyltransferases ST3GAL1 or ST6GALNAC1 to express homogeneously sT or sTn antigens. The level of BCG internalized was assessed by flow cytometry. The whole gene expression profile of BCG-challenged or unchallenged bladder cancer cell lines was studied by microarray technology. The profile of cytokines secreted by BCG-challenged bladder cancer cells and that of macrophages challenged by the secretome of BCG-challenged bladder cancer cells was studied by multiplex immune-beads assay. Results. Transcriptome analysis of the sialyltransferase-transduced cells revealed that groups of genes involved in specific functions were regulated in parallel in the two cell lines, regardless the sialyltransferase expressed. Namely, in sialyltransferase-expressing cells, genes involved in the proper chromosomal segregation and in the DNA repair were consistently down-regulated, while genes reported in literature as markers for bladder cancer were modulated. BCG-challenging induced a tendency to up-regulation of the genes preserving genomic stability and reducing malignancy, but only in cells expressing either sT or sTn. Among the ten cytokines tested, only IL-6 and IL-8 were expressed by bladder cancer cell lines and up-regulated by BCG-challenging, mainly in sialyltransferases-expressing cells. In macrophages, inflammatory cytokines, such as IL-1β, IL-6 and TNFα, and the antinflammatory IL-10 were induced only by the secretome of BCG-challenged bladder cancer cells, particularly when expressing either sialyltransferase, predicting the stimulation of M1-like macrophages and a better response to BCG therapy. Conclusions. The general effect of the expression of the two sialyltransferases and their products in the bladder cancer cells is toward a more malignant phenotype. However, the stronger ability of sialyltransferase expressing cells to produce inflammatory cytokines upon BCG-challenging and to stimulate macrophages predicts a more effective response to BCG in tumours expressing the sialylated TF antigens. This is fully consistent with our recent clinical data obtained in collaboration, showing that patients with bladder cancer expressing sTn respond better to BCG therapy.

Specific features of endothelial cells in primary tumors

RESUMO: As células endoteliais definem e delineiam todo o sistema vascular...Nesta tese procurámos explorar o papel que o ambiente tumoral exerce sobre as células endoteliais. ... Avaliamos também a capacidade anti-angiogénica de alguns derivados do estrogénio... Em suma os nossos resultados mostram a importância de um controlo rigoroso da regulação transcricional...

The influence of menopausal status on bone turnover and disease control in breast cancer patients with metastatic bone disease treated with chemotherapy plus zoledronic acid : an exploratory retrospective cohort study

RESUMO: Introdução: Uma meta-análise recente demonstrou que uso adjuvante de ácido zoledrónico (AZ) em mulheres pós-menopáusicas com cancro da mama precoce (CM) conduz a redução do risco de morte por CM em 17%. Investigámos o efeito do estado hormonal (pré [PrM] vs pós-menopausa tardia [PoM]) na remodelação óssea e controlo de doença em mulheres com CM e metástases ósseas (MO) tratadas com AZ e quimioterapia (QT). Métodos: Neste estudo de coorte retrospetivo, colhemos variáveis clinico-patológicas e quantificámos o telopéptido N-terminal (NTX) urinário e marcadores tumorais (MT) séricos em mulheres com CM e MO tratadas com QT e AZ. As doentes foram divididas em PrM (<45 anos) e PoM (>60 anos). Endpoints do estudo: variação do NTX, CA15.3 e CEA nos meses 3, 6 e 9, tempo até falência de QT de primeira-linha e sobrevivência. Quando apropriado foram usados os testes de Wilcoxon rank-sum, modelo de efeitos lineares mistos, teste log-rank e modelo de Cox. Resultados: Quarenta doentes foram elegíveis para análise (8 PrM e 32 PoM). Depois da introdução de AZ e QT, os níveis de NTX e MT caíram no coorte global. O perfil de resposta não diferiu entre grupos no mês 3 ou em tempos posteriores (valor-p para interação tempo-estado hormonal no mês 3=0.957). Ademais, o perfil de resposta dos MT também não diferiu entre grupos. O tempo mediano até falência de primeira-linha de QT em PrM e PoM foi de 15.2 e 17.4 meses, respetivamente. Não foi identificada diferença significativa entre grupos, quer em análise univariada quer após controlo para envolvimento visceral (p=0.399 e 0.469, respetivamente). Igualmente, não houve diferenças em termos de sobrevivência. Conclusões: Neste coorte, não foram identificadas diferenças no controlo de NTX ou MT em função do estado menopausico. Igualmente, não foi identificada diferença no tempo até falência de primeira-linha de CT ou sobrevivência.----------- ABSTRACT: Background: A recent meta-analysis showed that the adjuvant use of zoledronic acid (ZA) in postmenopausal women with early breast cancer (BC) leads to a reduction in the risk of breast cancer death by 17%. We investigated the effect of the hormonal status (pre [PrM] vs late post menopause [PoM]) on bone turnover and disease control among women with BC and boné metastases (BM) treated with ZA and chemotherapy (CT). Methods: In this retrospective cohort study, we collected clinicopathologic variables, urinary Nterminal telopeptide (NTX) and serum tumor marker levels from women with BC and BM treated with CT and ZA. Patients were divided in PrM (<45 years) and PoM (>60 years). Study endpoints were NTX, CA15.3 and CEA variation at 3, 6 and 9 months, and time to first-line CT failure and survival. We performed multilevel mixed-effects linear regression models to assess the variation of repeated measures and cox regression models for time to event outcomes. Results: Forty patients were eligible for analysis (8 PrM and 32 PoM). After introduction of ZA and CT, NTX and tumor markers declined in the overall cohort. Response profile was similar between menopausal groups at month 3 and at later time points (p-value for time-hormonal status interaction at month 3=0.957). Furthermore, tumor markers response profile was also equal between groups. Median time to first-line CT failure in PrM and PoM women was 15.2 and 17.4 months, respectively. No significant difference between groups was found, either using a univariate analysis or after controlling for visceral disease involvement (p=0.399 and 0.469, respectively). Likewise, no differences in survival were found. Conclusions: In this cohort, no differences were found in terms of NTX or tumor markers control according to menopausal status. Similarly, no difference in time to first-line CT failure or survival was found.

G-quadruplex ligands for cancer therapy

DNA may fold into a diversity of structures and topologies such as duplexes and triplexes. Some specific guanine-rich DNA sequences may even fold into a higher order structures denominated guanine G-quadruplexes (G4). These G-quadruplex forming sequences have shown biological interest since were found in telomeres and in promoter region of oncogenes. Thus, these G4 forming sequences have been explored as therapeutic targets for cancer therapy, since G4 formation was demonstrated to inhibit RNA-polymerase and telomerase activity. However, the G4 structures are transient and are only formed under specific conditions. Hence the main objective of this work is to develop new G4-specific ligands which may potentially find applications in the therapeutic area. Several potential G4-binding ligands were synthesized and characterized. The synthesis of these compounds consisted on a procedure based on van Leusen chemistry and a cross-coupling reaction through C-H activation, affording phenanthroline compounds (Phen-1, 50%; Phen-2, 20%), phenyl (Iso-1, 61%; Iso-2, 21%; Ter-1, 85%; Ter-2, 35%), and quinolyl (Quin-1, 85%; Quin-2, 45%) compounds. Screening assays for selecting the potential G4 compounds were performed by FRET-melting, G4-FID, CD-melting and DSF. Qualitative biophysical studies were performed by fluorescence and CD spectroscopy. Two high-specific G-quadruplex ligands, Phen-1 and Phen-2, were found to effectively bind telomeric and c-myc G4 structures. Phen-1 was found to stabilize parallel telomeric 22AG and c-myc sequence by 4.1 and 4.3 ˚C, respectively. Phen-2 also displayed high affinity towards 22AG (

Epstein-Barr virus-positive gastric cancer: a distinct molecular subtype of the disease?

Abstract: Approximately 90% of the world population is infected by Epstein-Barr virus (EBV). Usually, it infects B lymphocytes, predisposing them to malignant transformation. Infection of epithelial cells occurs rarely, and it is estimated that about to 10% of gastric cancer patients harbor EBV in their malignant cells. Given that gastric cancer is the third leading cause of cancer-related mortality worldwide, with a global annual incidence of over 950,000 cases, EBV-positive gastric cancer is the largest group of EBV-associated malignancies. Based on gene expression profile studies, gastric cancer was recently categorized into four subtypes; EBV-positive, microsatellite unstable, genomically stable and chromosomal instability. Together with previous studies, this report provided a more detailed molecular characterization of gastric cancer, demonstrating that EBV-positive gastric cancer is a distinct molecular subtype of the disease, with unique genetic and epigenetic abnormalities, reflected in a specific phenotype. The recognition of characteristic molecular alterations in gastric cancer allows the identification of molecular pathways involved in cell proliferation and survival, with the potential to identify therapeutic targets. These findings highlight the enormous heterogeneity of gastric cancer, and the complex interplay between genetic and epigenetic alterations in the disease, and provide a roadmap to implementation of genome-guided personalized therapy in gastric cancer. The present review discusses the initial studies describing EBV-positive gastric cancer as a distinct clinical entity, presents recently described genetic and epigenetic alterations, and considers potential therapeutic insights derived from the recognition of this new molecular subtype of gastric adenocarcinoma.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

Optimization of production of anti-cancer vaccines based on dendritic cells

Cancer remains as one of the top killing diseases in first world countries. It’s not a single, but a set of various diseases for which different treatment approaches have been taken over the years. Cancer immunotherapy comes as a “new” breath on cancer treatment, taking use of the patients’ immune system to induce anti-cancer responses. Dendritic Cell (DC) vaccines use the extraordinary capacity of DCs’ antigen presentation so that specific T cell responses may be generated against cancer. In this work, we report the ex vivo generation of DCs from precursors isolated from clinical-grade cryopreserved umbilical cord blood (UCB) samples. After the thawing protocol for cryopreserved samples was optimized, the generation of DCs from CD14+ monocytes, i.e., moDCs, or CD34+ hematopoietic stem cells (HSCs), i.e, CD34-derived DCs, was followed and their phenotype and function evaluated. Functional testing included the ability to respond to maturation stimuli (including enzymatic removal of surface sialic acids), Ovalbumin-FITC endocytic capacity, cytokine secretion and T cell priming ability. In order to evaluate the feasibility of using DCs derived from UCB precursors to induce immune responses, they were compared to peripheral blood (PB) moDCs. We observed an increased endocytosis capacity after moDCs were differentiated from monocyte precursors, but almost 10-fold lower than that of PB moDCs. Maturation markers were absent, low levels of inflammatory cytokines were seen and T cell stimulatory capacity was reduced. Sialidase enzymatic treatment was able to mature these cells, diminishing endocytosis and promoting higher T cell stimulation. CD34-derived DCs showed higher capacity for both maturation and endocytic capacity than moDCs. Although much more information was acquired from moDCs than from CD34-derived DCs, we conclude the last as probably the best suited for generating an immune response against cancer, but of course much more research has to be performed.

Colorectal cancer screening

Colorectal cancer (CRC) is the third most common cancer in the world, and mortality has remained the same for the past 50 years, despite advances in diagnosis and treatment. Because significant numbers of patients present with advanced or incurable stages, patients with pre-malignant lesions (adenomatous polyps) that occur as result of genetic inheritance or age should be screened, and patients with long-standing inflammatory bowel disease should undergo surveillance. There are different risk groups for CRC, as well as different screening strategies. It remains to be determined which screening protocol is the most cost-effective for each risk catagory. The objective of screening is to reduce morbidity and mortality in a target population. The purpose of this review is to analyze the results of the published CRC screening studies, with regard to the measured reduction of morbidity and mortality, due to CRC in the studied populations, following various screening procedures. The main screening techniques, used in combination or alone, include fecal occult blood tests, flexible sigmoidoscopy, and colonoscopy. Evidence from the published literature on screening methods for specific risk groups is scanty and frequently does not arise from controlled studies. Nevertheless, data from these studies, combined with recent advances in molecular genetics, certainly lead the way to greater efficacy and lower cost of CRC screening.

Phage display as a tool for generation of bio-therapeutic agents for breast cancer

Notch is a conserved signalling pathway, which plays a crucial role in a multiple cellular processes such as stem cell self-renewal, cell division, proliferation and apoptosis. In mammalian, four Notch receptors and five ligands are described, where interaction is achieved through their extracellular domains, leading to a transcription activation of different target genes. Increased expression of Notch ligands has been detected in several types of cancer, including breast cancer suggesting that these proteins represent possible therapeutic targets. The goal of this work was to generate quality protein targets and, by phage display technology, select function-blocking antibodies specific for Notch ligands. Phage display is a powerful technique that allows the generation of highly specific antibodies to be used for therapeutics, and it has also proved to be a reliable approach in identifying and validating new cancer-related targets. Also, we aimed at solving the tri-dimensional structure of the Notch ligands alone and in complex with selected antibodies. In this work, the initial phase focused on the optimization of the expression and purification of a human Delta-like 1 ligand mutant construct (hDLL1-DE3), by refolding from E. coli inclusion bodies. To confirm the biological activity of the produced recombinant protein cellular functional studies were performed, revealing that treatment with hDLL1-DE3 protein led to a modulation of Notch target genes. In a second stage of this study, Antibody fragments (Fabs) specific for hDLL1-DE3 were generated by phage display, using the produced protein as target, in which one good Fab candidate was selected to determine the best expression conditions. In parallel, multiple crystallization conditions were tested with hDLL1-DE3, but so far none led to positive results.

Microsatellite instability in solitary and sporadic gastric cancer

Recently, the presence of microsatellite instability (MSI) has been reported in gastric cancer and associated with older age of presentation, distal tumor location, early disease staging, and better overall prognosis. Different characteristics in presentation and in tumor behavior may be explained by different genetic alterations during carcinogenesis of gastric cancer. Identification of specific genetic pathways in gastric cancer may have direct impact on prognosis and selection of treatment strategies. PATIENTS AND METHODS: All 24 patients were treated by radical surgery. Fragments of normal and tumor tissues were extracted from the specimen and stored at -80ºC before DNA purification and extraction. PCR amplification utilizing microsatellite markers was performed. Tumors presenting PCR products of abnormal sizes were considered positive for microsatellite instability (MSI+). RESULTS: Five patients (21%) had tumors that were MSI+ in at least 1 marker. In the group of patients with Lauren's intestinal-type gastric carcinoma, 3 had tumors that were MSI+ (23%), while in the group of diffuse-type gastric cancer, 2 patients had tumors that were MSI+ (19%). The mean age of presentation and the male:female ratio was similar in both groups. Tumors that were MSI+ were more frequently located in proximal portion of the stomach compared to microsatellite-stable (MSS) tumors (40% vs. 16%). Although there was a trend of patients with MSI+ tumors towards a proximal gastric tumor location, early staging, and negative lymph node metastasis, there was no statistical significance compared to those with MSS tumors (P >.1). Comparison of overall and disease-free survival between gastric tumors that were MSI+ and those that were MSS found no statistically significant differences (P >.1). CONCLUSIONS: Microsatellite instability is a frequent event in gastric carcinogenesis and shows a trend towards distinct clinical and pathological characteristics of gastric cancer.

Therapeutics bioconjugates: evaluation of iminoboronates as payload delivery system for cancer

The advent of bioconjugation impacted deeply the world of sciences and technology. New biomolecules were found, biological processes were understood, and novel methodologies were formed due to the fast expansion of this area. The possibility of creating new effective therapies for diseases like cancer is one of big applications of this now big area of study. Off target toxicity was always the problem of potent small molecules with high activity towards specific tumour targets. However, chemotherapy is now selective due to powerful linkers that connect targeting molecules with affinity to interesting biological receptors and cytotoxic drugs. This linkers must have very specific properties, such as high stability in plasma, no toxicity, no interference with ligand affinity nor drug potency, and at the same time, be able to lyse once inside the target molecule to release the therapeutic warhead. Bipolar environments between tumour intracellular and extracellular medias are usually exploited by this linkers in order to complete this goal. The work done in this thesis explores a new model for that same task, specific cancer drug delivery. Iminoboronates were studied due to its remarkable selective stability towards a wide pH range and endogenous molecules. A fluorescence probe was design to validate this model by creating an Off/On system and determine the payload release location in situ. A process was optimized to synthetize the probe 8-(1-aminoethyl)-7-hydroxy-coumarin (1) through a reductive amination reaction in a microwave reactor with 61 % yield. A method to conjugate this probe to ABBA was also optimized, obtaining the iminoboronate in good yields in mild conditions. The iminoboronate model was studied regarding its stability in several simulated biological environments and each half-life time was determined, showing the conjugate is stable most of the cases except in tumour intracellular systems. The construction of folate-ABBA-coumarin bioconjugate have been made to complete this evaluation. The ability to be uptaken by a cancer cell through endocytosis process and the conjugation delivery of coumarin fluorescence payload are two features to hope for in this construct.

Logic programming and artificial neural networks in breast cancer detection

About 90% of breast cancers do not cause or are capable of producing death if detected at an early stage and treated properly. Indeed, it is still not known a specific cause for the illness. It may be not only a beginning, but also a set of associations that will determine the onset of the disease. Undeniably, there are some factors that seem to be associated with the boosted risk of the malady. Pondering the present study, different breast cancer risk assessment models where considered. It is our intention to develop a hybrid decision support system under a formal framework based on Logic Programming for knowledge representation and reasoning, complemented with an approach to computing centered on Artificial Neural Networks, to evaluate the risk of developing breast cancer and the respective Degree-of-Confidence that one has on such a happening.

Monocarboxylate transport inhibition potentiates the cytotoxic effect of 5-fluorouracil in colorectal cancer cells

Cancer cells rely mostly on glycolysis to meet their energetic demands, producing large amounts of lactate that are extruded to the tumour microenvironment by monocarboxylate transporters (MCTs). The role of MCTs in the survival of colorectal cancer (CRC) cells is scarce and poorly understood. In this study, we aimed to better understand this issue and exploit these transporters as novel therapeutic targets alone or in combination with the CRC classical chemotherapeutic drug 5-Fluorouracil. For that purpose, we characterized the effects of MCT activity inhibition in normal and CRC derived cell lines and assessed the effect of MCT inhibition in combination with 5-FU. Here, we demonstrated that MCT inhibition using CHC (a-cyano-4-hydroxycinnamic acid), DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and quercetin decreased cell viability, disrupted the glycolytic phenotype, inhibited proliferation and enhanced cell death in CRC cells. These results were confirmed by specific inhibition of MCT1/4 by RNA interference. Notably, we showed that 5-FU cytotoxicity was potentiated by lactate transport inhibition in CRC cells, either by activity inhibition or expression silencing. These findings provide novel evidence for the pivotal role of MCTs in CRC maintenance and survival, as well as for the use of these transporters as potential new therapeutic targets in combination with CRC conventional therapy.