Notch receptors and ligands are type I transmembrane proteins that regulate development and differentiation during cell-cell contact. There are four Notch receptor homologues and five notch ligands, identified in humans till date. Upon ligand activation, Notch1 intracellular domain (NIC-1) is released into the cytoplasm, which binds to several proteins as well as translocates into the nucleus to effect the Notch signaling. In the absence of the activated Notch signaling, the Notch target genes are kept repressed by the transcriptional repressor C protein binding factor 1 (CBF1) also known as RBPjk or CSL for CBF1/Su(H)/Lag1. RBPjk binds to the sequence “CGTGGGAA” and acts as a constitutive repressor. Upon ligand dependent activation, NIC-1 enters into the nucler and converts RBPjk from transcriptional repressor to an activator. Notch binding to CSL replaces the SMRT corepressor complex with a coactivator complex including SKIP, Mastermind like 1 (MAML1) (Mastermind in Drosophila), and histone acetyl transferases PCAF, GCN5 and p300 activating the transcription of target genes. Mastermind-like (MAML), a family of transcriptional activator proteins comprising of 3 members 1 to 3, has been shown to be required for Notch signaling. MAML forms a ternary complex with RBPjk-NIC by directly interacting with NIC. In turn, MAML recruits the histone acetyl transferase p300/CBP, which acetylates the histones, thereby altering the structure of chromatin amenable for transcription. Activation of Notch pathway induces oncogenesis, which can be divided into two categories including 1) Inhibition of Apoptosis and 2) Induction of proliferation. In T cells, activation of Notch1 protects cells from T cell receptor, dexamethasone and etoposide-mediated apoptosis, Fas receptor-mediated signaling by up regulating IAP (Inhibitor of Apoptosis) and Bcl-2 families, as well as FLIP (FLICE-like inhibitor protein). Notch signaling also promotes the survival of T cells through maintenance of cell size as well as through the promotion of glucose uptake and metabolism. Notch-1 has been shown to protect against anoikis (apoptosis induced by matrix withdrawal) or p53-mediated apoptosis in immortalized epithelial cells, T cell receptor-induced apoptosis in mature cells and dexamethasone-mediated apoptosis in thymocytes. This study was carried out to functionally characterize NIC-1 (human Notch1-intracellular domain) as an inhibitor of apoptosis and to evaluate the therapeutic potential of reversal of this apoptosis inhibition. The main objectives of this study are 1. Construction of recombinant adenovirus expressing human Notch1-intracellular domain (Ad-NIC-1) and characterization of NIC-1 as an inhibitor of chemotherapy and p53-induced cytotoxicity and apoptosis. 2. Role of PI3 kinase -Akt/PKB -mTOR pathway in NIC-1-mediated inhibition of p53-induced apoptosis. 3. Essential role of association between mTOR and NIC-1 and the dependent NIC-1 phosphorylation in Notch1-mediated transcription and survival signaling. 4. Identification of NIC-1 as an inhibitor of E1A-induced apoptosis and the role of mTOR in NIC-1-mediated inhibition of E1A-induced apoptosis. Activated Notch1 was first linked to tumorigenesis through identification of a recurrent t(7;9)(q34;q34.3) chromosomal translocation involving the human Notch1 gene that is found in a subset of human pre-T-cell acute lymphoblastic leukemia’s (T-ALL). Deregulated Notch signaling is oncogenic, inhibits apoptosis and promotes survival. In order to understand survival signaling induced by Notch1 and its possible role in chemoresistance, we have generated a replication deficient recombinant adenovirus expressing human Notch1-intracellular domain (Ad-NIC-1) and shown that it produces functional NIC-1 protein. Using this overexpression system, we characterized that activated Notch1-inhibits chemotherapy and in particular p53 induced apoptosis. Notch1-mediated inhibition of p53-induced apoptosis does not include coactivator squelching. p53 was inefficient in binding to its DNA in NIC-1 overexpressing cells. The levels of phosphorylation at Ser15, Ser20, and Ser392 of p53 expressed from Ad-p53 significantly reduced in NIC-1 preinfected cells. These results suggest that NIC-1-mediated inhibition of p53-mediated apoptosis involves reduced DNA binding, reduced nuclear localization and reduced post translational modifications and thus reduced transactivation of its target genes. Notch1-mediated inhibition of p53 was found to occur mainly through mammalian target of rapamycin (mTOR) using PI3 kinase-Akt/PKB pathway, as the mTOR inhibitor; rapamycin treatment was able to reverse Notch-1 mediated inhibition of p53 and chemoresistance. Consistent with this, rapamycin failed to reverse NIC-1 induced chemoresistance in cells expressing rapamycin resistant mTOR. Our results also suggest that the N-terminal HEAT repeat and the kinase function of mTOR are essential for Notch mediated inhibition of p53. Further, ectopic expression of eIF4E, a translational regulator that acts downstream of mTOR, inhibited p53-induced apoptosis and conferred protection against p53-mediated cytotoxicity to similar extent as that of NIC-1 overexpression, but was not reversed by rapamycin, which indicates that eIF4E is the major target of mTOR in Notch1-mediated survival signaling. Notch1-intracellular domain (NIC-1), following proteolytic cleavage, binds to RBPjk and regulates transcription. Active NIC-1 located in the nucleus is phosphorylated, which makes it more stable and bind better to RBPjk. NIC-1 was also shown to bind to Deltex1 in the cytoplasm. Next, we studied the requirement of components of Notch1 signaling pathway for this function. By using variety of approaches, we found that both RBPjk and Maml1 and hence transcription activation is required for NIC-1-mediated survival signaling and inhibition of p53 functions. Interestingly, while we found the other Notch1 effector, Deltex1 is also required for above functions, Notch1 failed to activate PI3 kinase -Akt/PKB -mTOR pathway in Deltex1, but not in RBPjk silenced cells. Our results suggest that Notch-Deltex1 pathway activates PI3 kinase. Previous studies show that NIC-1 interacts with Deltex1 and Grb2 interacts with PI3 kinase. Our data shows that Deltex1 interacts with SH3 domain of Grb2. Since Notch1-Deltex1 and PI3 kinase-Grb2 interactions are known, we conclude that Notch1 activation of PI3 kinase involves Deltex1 and Grb2. We found activated mTOR was able to binds to NIC-1 and regulates its phosphorylation. Inhibition of mTOR either by PI3 kinase inhibitors or mTOR inhibitor treatment or silencing of Akt/PKB or mTOR reduced the phosphorylation of NIC-1 with the concomitant reduction in NIC-1-mediated transcription. Further, endogenous Notch1 receptor activated by the DSL ligand failed to activate transcription efficiently in rapamycin treated cells, implying a positive role for mTOR in mammalian Notch signaling. These studies reveal that Notch1 activates PI3 kinase -Akt/PKB -mTOR signaling through Deltex1 and subsequently activated mTOR modulates Notch1 signaling by direct binding and possibly thorough phosphorylation of the intracellular domain of Notch. Adenoviral E1A, in the absence of cooperating oncogene, suppresses primary tumor growth and reverses the transformed phenotype of human tumor cells by inducing apoptosis. E1A requires p53 for efficient induction of apoptosis and was shown to induce apoptosis by down regulating Akt and the activation of pro apoptotic factor p38 MAP kinase. Since our results suggest Notch1 inhibits chemotherapy and p53-induced apoptosis, we analyzed the ability of Notch1 to protect cells from E1A-induced apoptosis. Here we show that NIC-1 suppresses the ability of E1A to induce apoptosis. NIC-1 requires mTOR-dependent signal to inhibit E1A-mediated apoptosis, as the rapamycin, an mTOR inhibitor was able to completely reverse the ability of Notch1 to protect cells against E1A-induced apoptosis. The role of mTOR in NIC-1-mediated survival signaling was further confirmed by using the cells stably expressing rapamycin resistant mTOR. Rapamycin was able to reverse Notch1-mediated protection in cells expressing wild type mTOR but not in rapamycin resistant mTOR expressing cells. We also found that E1A was able to induce apoptosis in cells silenced for the pro apoptotic factor p38 and NIC-1 continued to inhibit E1A-induced apoptosis in these cells. These results confirm that Notch1 requires the activation of mTOR signaling but not p38 MAP kinase for inhibition of E1A-induced apoptosis. These results also suggest that the combination therapy utilizing E1A-mediated gene delivery in combination with inhibition of mTOR pathway may prove successful in treating Notch overexpressing cancers. Chemotherapy remains a major treatment modality for human cancers. Chemoresistance is a clinical problem that severely limits treatment success. It can be divided into two forms: intrinsic and acquired. Intrinsic resistance is the essence of oncogenic transformation, resulting from activation of oncogenes and the loss of tumor suppressors, and manifests itself as alterations in cell cycle checkpoints and apoptotic pathways. It is now widely accepted that the apoptotic capacity of the cancer cell is crucial in determining the response to chemotherapeutic agents. Indeed, several gene products that regulate apoptosis, i.e., p53, Akt and PI3K are frequently altered in cancer cells. In this study, we identified that cells with aberrant Notch1 signaling are chemoresistant. Activated Notch1 overexpression makes cells resistant to chemotherapy in a wild type p53 dependent manner. Notch protected p53 wild type cells but not p53 mutated or p53 deleted cells against chemotherapy induced cytotoxicity. Further, inactivation of p53 by specific silencing abrogated the ability of NIC-1 to protect H460 cells against adriamycin induced cytotoxicity. Most importantly, NIC-1 mediated chemoresistance can be reversed by blocking PI3 kinase -Akt/PKB -mTOR pathway. Collectively, these results suggest that cancers with activated Notch1 signaling are chemoresistant and provide basis for the reversal of chemoresistance.

Recently HER3, member of the epidermal growth factor receptor family (EGFR), has been found to play a crucial role in the development of resistance towards inhibitors that are given to patients with HER1- and HER2-driven cancers. As HER3 is up-regulated or over-activated in several types of human cancers, it is of outmost importance that new innovative drugs target its oncologic activity. The Affibody® Molecule Z08698 inhibits the heregulin induced signalling of HER3 with high affinity (KD~50 pM). As the Affibody® Molecule is small, has high solubility and outstanding folding kinetics, an effective penetration of tumour tissue is suggested together with a rationalized manufacturing process. Further coupling to an albumin binding domain (ABD) expands the plasma half-life of the molecule, hence increasing the molecule's potential of serving as a therapeutic. A process development for production of Z08698-VDGS-ABD094 has been established, where the molecule is efficiently produced in the E. coli host strain BL21(DE3), through a T7 based expression system. Cultivations were performed with a fed-batch fermentation process and the conditions were further optimized in order to obtain highest expression, while avoiding undesirable modifications like gluconoylations. By employing Design of experiments in combination with multivariate data analysis, a production process resulting in ~3.5 g product/ l culture could be verified. Moreover, thermolysis was evaluated as a suitable method for cell disruption, enabling an easy and cost-effective manufacturing process of the ABD fused Affibody® Molecule.

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No. of bitstreams: 2 Tese - Luis Cesar Branquinho - 2014.pdf: 6091709 bytes, checksum: 2c59441af9866c02cd7a2cc3cc667b3e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-12-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Magnetic nanoparticles can generate heat when submitted to alternating magnetic fields of adequate amplitude and frequency. This phenomenon is named magnetic hyperthermia and has several therapeutic applications, as for example, in the treatment of cancer. In general, the theoretical models used to describe this neglect the effect of interparticle interaction. In this thesis we investigate the effect of magnetic dipolar interaction in the magnetothermal efficiency (named specific loss power – SLP) of bicompatible magnetic nanoparticles. Firstly, we develop a chain of magnetic particles model, where we prove that the interaction leads to a contribution to the uniaxial anisotropy. This term in the free energy density allowed us to extract from the electron magnetic resonance technique (EMR) information about the mean chain size in the colloid. Further, this additional magnetic nanoparticle anisotropy term was used to develop an analytical theoretical model that takes into account the effect of the dipolar interaction between nanoparticles to SLP, considering the case where the magnetization responds linearly to the field (Linear Response Theory). Our calculations indicate that depending on the particle parameters, specially the anisotropy, the effect can be to enhance or decrease the heat generation. Moreover, we showed that increasing the chain size (number of particles in the chain) the optimal particle size for hyperthermia can decrease up to 30% in comparison with non-interacting particles. This result has several clinical implications, which allowed us to suggest some strategies for improving the therapeutic efficacy. In order to investigate experimentally the effect, two magnetic fluids, one containing spherical nanoparticles based on manganese ferrite (MnF-citrate) in the superparamagnetic regime, and another commercial one (BNF-starch) magnetite-based with a shape of a parallellepiped and blocked, were selected and deeply characterized. We found a decrease of SLP increasing the chain size for the MnF sample, while for BNF-starch no effect was found at the same experimental conditions. The decrease of SLP in the MnF sample, within the particle concentration range, was explained considering in the model not only the effect in the anisotropy but also by an increase in the damping factor parameter, a term correlated to spin-phonon interaction. Data obtained using EMR and Monte Carlo simulations corroborate our hypothesis. The absence of concentration effect for the BNF sample was attributed to the higher anisotropy value and to the probable influence of brownian relaxation. In addition, the same chain model was used to investigate the behavior of blocked nanoparticles of Stoner-Wohlfarth type. In this case, we demonstrate that the chain formation increases the magnetic hyperthermia, as found in magnetosomes. Finally, we showed that a fluctuation of the dipolar interaction field between particles in the chain, which does not destroy the symmetry of this term, shows a Vogel-Fulcher behaviour in the weak coupling regime. / Nanopartículas magnéticas são capazes de gerar calor quando submetidas a campo magnético alternado de amplitude e frequência adequadas. Este fenômeno é conhecido como magnetohipertermia e possui aplicações terapêuticas como, por exemplo, no tratamento de câncer. Em geral, os modelos teóricos que descrevem o fenômeno não levam em conta efeitos associados à interação partícula-partícula. Nesta tese investigamos o efeito da interação dipolar magnética na eficiência magnetotérmica (SLP) de nanopartículas magnéticas biocompatíveis. Primeiramente desenvolvemos um modelo de cadeia de nanopartículas magnéticas, aonde provamos que a interação entre partículas que formam uma cadeia linear equivalem a uma contribuição uniaxial a anisotropia. Essa contribuição à densidade de energia permitiu que obtivéssemos por meio da técnica de ressonância magnética eletrônica (RME) informações acerca do tamanho médio de aglomerado na suspensão coloidal. Posteriormente utilizamos esse termo adicional da anisotropia efetiva da nanopartícula para propor um modelo teórico analítico que leve em consideração o efeito de tal interação na eficiência de aquecimento de nanopartículas magnéticas em um fluido para o caso em que a magnetização das nanopartículas responde linearmente ao campo (Teoria da Resposta Linear). Nossos cálculos indicaram que, dependendo de parâmetros da nanopartícula, em particular da anisotropia, este efeito pode aumentar ou diminuir a geração de calor. Além disso, mostramos que o aumento do número de partículas formando cadeias lineares reduz o diâmetro ótimo para hipertermia em até 30% em relação ao valor esperado para partículas isoladas. Este resultado possui fortes implicações clínicas, e permitiu que sugeríssemos algumas estratégias para aumentar a eficiência terapêutica. No intuito de investigar experimentalmente este efeito, dois fluidos magnéticos, um contendo nanopartículas esféricas de ferrita de Mn (MnF-citrato) no regime superparamagnético e outra comercial (BNF-starch) à base de magnetita com forma de nanoparalelepípedos e contendo partículas bloqueadas, foram selecionados e amplamente caracterizados. Observamos uma diminuição no SLP com o aumento de partículas na cadeia para a amostra MnF-citrato, para todos os valores de campo, enquanto que para a amostra BNF-starch não percebemos alteração do SLP. O decréscimo do SLP da amostra MnF, na faixa de concentração investigada, foi explicado incluindo não apenas o efeito na anisotropia efetiva, mas também o aumento no valor do fator de amortecimento. Dados de RME e simulação de Monte Carlo corroboraram tal hipótese. A ausência de efeito para amostra BNF-starch foi atribuída à alta anisotropia e provável influência de relaxação browniana. Adicionalmente, o modelo de cadeia foi usado para explicar o comportamento de nanopartículas bloqueadas do tipo Stoner-Wohlfarth. Neste caso demonstramos que a formação de cadeias aumenta a hipertermia magnética, como verificado em magnetossomos. Finalmente, mostramos que uma flutuação no campo dipolar interpartículas na cadeia, que não destrua a simetria desta contribuição, fornece um comportamento do tipo Vogel-Fulcher no regime fracamente interagente.

Les peptides pénétrants sont de petits peptides capables de se rentrer dans les cellules sans endommager la membrane, présentant un grand potentiel dans la délivrance de diverses cargaisons, y compris des peptides, pour le traitement du cancer ainsi que d'autres maladies. Les peptides comme médicaments, bénéficient d'être spécifiques, relativement sûrs, faciles à produire et faciles à modifier. Cependant, des défis significatifs demeurent concernant l'application de peptides en tant qu'agents thérapeutiques. L'identification des motifs de liaison des peptides est difficile et les peptides se comportent généralement avec une perméabilité cellulaire faible et une sensibilité à l'hydrolyse des proteases. Dans le présent travail, nous avons caractérisé deux interactions protéine-protéine Ras/Raf et PP2A/SET qui sont impliqués dans la régulation de la transformation tumorale et de l'apoptose. Nous avons identifié le site de liaison parmi ces protéines (peptides interférents). Ces peptides interférents ont été associés à une navette optimisée pour générer des peptides chimériques capables de dissocier ces interactions protéine/protéine. Les peptides chimeriques ont été testés in vitro et in vivo, montrant un effet anti-tumor. Ces peptides pourraient être considérés comme des candidats prometteurs pour des applications futures en tant que vecteurs pour la délivrance de médicaments intracellulaires. / Cell penetrating peptides are small peptides which are able to translocate into cells without causing membrane damage, presenting a great potential in the delivery of various cargos including peptides, for the treatment of cancer as well as other diseases. Peptides as drugs, benefit from being specific, relative safe, easy to produce and easy to modify. However, significant challenges remain regarding the application of peptides as therapeutic agents. Identification of the binding motifs of the peptides is difficult, and peptides generally behave low-cellular permeability and sensitivity to proteases hydrolysis. In the present work, we characterized two protein-protein interactions Ras/Raf and PP2A/SET that are involved in the regulation of tumor transformation and apoptosis. We have identified the binding site among these proteins (interfering peptides). These interfering peptides were associated to an optimized shuttle to generate chimeric peptides able to dissociate these protein/protein interactions. The chimeric peptides were tested in vitro an in vivo, showing an anti-tumor effect. These peptides could be considered as promising candidates for future applications as vectors for intracellular drugs delivery.

La résistance aux médicaments constitue un obstacle majeur pour le traitement du cancer. Les systèmes nanoparticulaires de délivrance de médicaments (nanoparticule drug delivery system, NDDS) sont pressentis pour apporter un nouvel espoir dans le traitement du cancer afin de surmonter la résistance aux médicaments en délivrant spécifiquement l’agent anticancéreux dans la lésion tumorale par effet EPR. Cela aura pour effet d’augmenter la concentration locale en médicaments et par conséquent d’améliorer l'efficacité thérapeutique tout en épargnant les tissus sains afin d'éviter les effets secondaires liés à la thérapie. Dans la mesure où la tumeur a souvent un microenvironnement acide, nous souhaiterions en outre doter nos nanoparticules NDDS d’une sensibilité pH-dépendante afin de permettre une délivrance spécifique dans la tumeur. Au cours de cette thèse, nous avons élaboré différents NDDSs sensibles aux variations de pH en employant des stratégies différentes. Ces NDDSs peuvent spécifiquement libérer le médicament au niveau du tissu tumoral et dans les cellules elles-mêmes à des valeurs de pH acides. En augmentant la concentration intracellulaire de médicament, l'objectif de surmonter la résistance aux médicaments pourrait ainsi être atteint. La présente étude a permis de fournir de nouvelles connaissances sur la conception de nano-transporteurs pour surmonter la résistance multidrogue par l’élaboration de NDDS sensibles au pH et constitue donc un exemple illustrant parfaitement le fait que les progrès des nanotechnologies peuvent être avantageusement mis en œuvre pour développer de nouvelles perspectives thérapeutiques. / Drug resistance presents a great hurdle to cancer treatment. Nanotechnology-based drug delivery systems (NDDSs) are widely expected to bring new hope for cancer therapy to overcome drug resistance by specifically delivering anticancer drugs to tumor lesions via the EPR effect, hence increasing local drug concentrations and consequently enhancing therapeutic efficacy, and at the same time, sparing healthy tissues to avoid side effects. As tumors often have an acidic microenvironment, we would like to further endow the NDDS with a pH-responsive drug releasing property for specific tumor targeting. In this thesis, we established different pH-responsive NDDSs by employing different strategies. These NDDSs could specifically control drug release at tumor tissues and within tumor cells in response to acidic pH. By increasing the intracellular drug concentration, the goal of circumventing drug resistance in cancer was achieved. The present study provides new insights into the design of nanocarriers to overcome drug resistance through pH-responsive drug delivery, and illustrates how advances in nanotechnology can be advantageously implemented to enhance therapeutic outcomes.

Le cancer du poumon est la principale cause de décès par cancer chez les hommes et les femmes dans le monde. Malgré les avancées majeures dans les traitements, les thérapies à base de platine restent la thérapie standard pour les patients atteints du cancer du poumon induit par la mutation KRAS. Bien que les composés de platine aient un premier effet sur ces patients, l’apparition d’une rechute constitue le principal défi au niveau clinique.Dans ce contexte, notre projet actuel vise à caractériser l’activation de la voie Notch dans la réponse aux sels de platines d’adénocarcinomes mutés pour K-Ras ainsi que son rôle dans l’acquisition de la résistance à ce traitement. Nous avons mis en évidence que la voie Notch est activée en réponse à divers agents induisant des dommages à l’ADN dans des lignées de cancer du poumon. Nous avons montré que cette activation est dépendante de la voie de checkpoint de Réponse aux Dommages de l’ADN (DDR) via ATM, ATM qui est une des principales kinases de la réponse cellulaire aux lésions de l’ADN telles que les cassures double-brin, causé notamment par la chimiothérapie conventionnelle. Parmi les nombreux substrats d’ATM en réponse à ces dommages, nous montrons un effet direct de MDM2 dans l’activation de la voie Notch en réponse aux dommages à l’ADN causé par le carboplatine. Nos données montrent une nouvelle interaction entre ATM, MDM2 et Notch, lors du traitement au carboplatine, qui pourrait jouer un rôle dans la cascade de signalisation favorisant la survie des cellules tumorales pulmonaires.En utilisant des xénogreffes dérivées de patients atteints d’adénocarcinomes du poumon, ainsi qu’un modèle cellulaire de xénogreffe résistant à la carboplatine, nous avons montré que l’association d’inhibiteurs de Notch et de MDM2 associé au traitement au carboplatine ont un effet accru sur le ralentissement de la croissance de la tumeur et sur la survie, en comparaison avec le traitement actuel de référence que représente le carboplatine.Nos résultats offrent une nouvelle possibilité de thérapie pour les adénocarcinomes de poumons mutés pour KRAS, et pourrait donc répondre à un besoin clinique urgent notamment pour contrecarrer la résistance aux chimiothérapies conventionnelles à base de sels de platine. / Lung cancer is the leading cause of cancer death among men and women worldwide. Despite the major advances in the treatments, platinum-based therapy remains the standard of care for patients affected by KRAS-driven lung cancer. Even though the platinum-compounds display an initial effect on these patients, the onset of the relapse constitutes the main challenge for the clinic. The molecular mechanisms underlying lung adenocarcinoma (LUAD) relapse are not completely elucidated yet, thus it is fundamental to understand them in order to improve survival of patients. Our data show that upon carboplatin treatment, the Notch pathway is activated in vitro. Since this effect was common for several other DNA damage insults, our driving hypothesis connected the DNA Damage Response (DDR) to the activation of the Notch pathway. Our data demonstrates that protein kinase ataxia telangiectasia-mutated (ATM) is a key mediator in the activation of the Notch pathway during DNA damage signalling. ATM is well-known as the chief mobilizer of the cellular response to the most toxic lesions to the DNA, the double-strand breaks which are also the type of damage caused by the conventional chemotherapy. Among the several substrates of ATM in response to the damage, there is the mouse double minute 2 protein (MDM2) that it is known to interact with the Notch pathway. Our data uncovered a new pathway connecting ATM, MDM2 and NICD during carboplatin treatment in LUAD cells.Using LUAD Patient-Derived Xenografts and a new generated carboplatin resistant cellular model, we show in vivo that the combination of Notch and MDM2 inhibitors with carboplatin showed a therapeutic benefit in tumour growth and survival compared to the standard of care, i.e. carboplatin.Our results can offer a new therapeutic window for KRAS-driven LUAD that become resistant to platinum-based therapy, hence tackling an urgent and unmet clinical need.

<p>Vitamin E is a natural lipophilic antioxidant contains eight structurally related forms, i.e., α-, β-, γ-, δ-tocopherols (αT, βT, γT, and δT) and corresponding tocotrienols. Recent research indicates that vitamin E forms are differentially metabolized to various carboxychromanols. Some these vitamin E metabolites have been shown to exhibit strong anti-inflammatory and anticancer effects, yet little is known about their bioavailability. Without this knowledge, it is impossible to assess the role of vitamin E metabolism in biological functions of vitamin E forms and their protective effects on chronic diseases. While αT and γT appear to improved gut health, the underlying mechanisms are not well understood. Furthermore, specific forms of vitamin E such as γT have been reported to have cancer-preventing effects, but their anticancer efficacy is relatively modest. For these reasons, this dissertation focused on the characterization of the pharmacokinetic formation of vitamin E metabolites after supplementation, and the investigation of the underlying mechanisms of the protective effect of vitamin E forms, αT and γT, on gut health, as well as anticancer efficacy of the combination of aspirin and γT on carcinogen-induced colon tumorigenesis.</p><p><br></p><p>The first project focuses on characterizing the pharmacokinetic formation of vitamin E metabolites after single dose supplementation of γ-tocopherol-rich mixed tocopherol (γTmT) and δ-tocotrienol (δTE). With our recently developed LC/MS/MS assay for quantifying vitamin E metabolites, we can simultaneously quantify the level of short-chain, long-chain, and sulfated carboxychromanols in plasma, urine, and fecal samples of supplemented animals. In this study, we investigated the pharmacokinetics including excretion of vitamin E forms and the formation of their metabolites after a single dose intragastric administration of tocopherols and tocotrienols in rats. We also measured vitamin E metabolites in the serum obtained from healthy humans after gT supplementation. In the plasma of rat, the pharmacokinetic profiles of γT and δTE are described as the following: γT, Cmax = 25.6 ± 9.1 μM, Tmax = 4 h; δTE, Cmax = 16.0 ± 2.3 μM, Tmax = 2 h. Sulfated CEHCs and sulfated 11’-COOHs were the predominant metabolites in the plasma of rat with Cmax of 0.4-0.5 μM (Tmax ~ 5-7 h) or ~0.3 μM (Tmax at 4.7 h), respectively. In 24-h urine, 2.7% of γT and 0.7% of dTE were excreted as conjugated CEHCs, the major identified urinary metabolites. In the feces, 17-45% of supplemented vitamers were excreted as un-metabolized forms and 4.9-9.2% as metabolites. The majority of metabolites excreted in feces were unconjugated carboxychromanols, among which 13’-COOHs constituted ~50% of total metabolites. Interestingly, 13’-COOHs derived from δTE were 2-fold higher than 13’-COOH from γT. Unlike rats, γ-CEHC is the predominant metabolites found in human plasma, although 11’-COOHs and 13’-COOHs (sulfated and unconjugated) were elevated by >20 folds responding to γT supplement. In this study, we found that tocopherols and tocotrienols, when taken as supplements, are mainly excreted as un-metabolized forms and long-chain carboxychromanols in feces. High fecal availability of 13’-COOHs may contribute to modulating effects on gut health.</p><p><br></p><p>The second project of my dissertation investigated the effect of vitamin E forms, αT and γT, on intestinal barrier function in a cellular model and a mouse colitis model. Inflammatory bowel diseases (IBD) are chronic idiopathic inflammatory conditions characterized by disruption of intestinal barrier integrity. Previous studies by others and us had demonstrated that vitamin E forms, αT and γT, can protect against chemical-induced colitis in animal models. However, the role of these vitamin E forms on intestinal barrier function has not been studied. Herein, we investigated the potential protective effects of vitamin E forms, αT and γT, on intestinal barrier function in a Caco-2 colon epithelial cell model and a dextran sodium sulfate (DSS)-induced colitis mouse model. In Caco-2 cells, pretreatment with 25mM αT and γT attenuated Caco-2 monolayer barrier dysfunction induced by 10 ng/mL TNF-α/IFN-γ, suggesting that these vitamin E forms protect intestinal barrier integrity in this cellular model. In male BALB/c mice, the supplementation of αT (0.05%) or γTmT (0.05%) when given 3 weeks before DSS treatment or at the same time as DSS treatment alleviated DSS-induced fecal bleeding and diarrhea symptoms in mice, and attenuated colon inflammation and colitis-associated damages. Additionally, αT and γTmT supplementation attenuated DSS-induced intestinal barrier dysfunction, as indicated by improving the level of occludin, a tight junction protein, in the colon and reducing lipopolysaccharide-binding protein (LBP) in the plasma. Furthermore, gut microbiota analysis demonstrated that αT and γTmT supplementation could modulate intestinal microbiome composition in mice with DSS treatment. DSS treatment reduced the relative abundance of Lachnospiraceae compared to healthy mice, and supplementation of αT and γT partially reversed this effect. Interestingly, the family Lachnospiraceae has been reported to decrease in IBD patients. Our study demonstrated the protective effects of vitamin E forms on intestinal barrier integrity in a cell-based model and a colitis model in mice. Furthermore, we demonstrated that these vitamin E forms caused favorable changes in the intestinal microbial population under colitis condition.</p><p><br></p><p>The third project of my dissertation evaluated the anticancer efficacy of the combination of aspirin and γT using an azoxymethane (AOM)-induced and colitis-promoted colon tumorigenesis mouse model. Extensive inflammation in the colon promotes the development of colorectal cancer (CRC). Eicosanoid production by pro-inflammatory enzymes, cyclooxygenases (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) play a critical role in the initiation, progression, and invasion of CRC. Thus, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, have been recommended for chemoprevention of CRC. However, long-term use of aspirin can cause many side effects, and the anticancer activity of aspirin is very modest. Previously, we have demonstrated that the combination of γT with aspirin prolonged the anti-inflammatory activity of aspirin and alleviated aspirin-associated adverse effects in a carrageenan-induced inflammation model in rats. Additionally, we found that the combination of γT and aspirin has stronger anticancer activity than aspirin or γT alone against HCT-116 human colorectal carcinoma cells. Therefore, we examined the anticancer effect of the combination of 0.025% aspirin and 0.05% γT against AOM-induced and DSS-promoted tumorigenesis in mice. In this study, we have found that the combination of aspirin and γT, but not aspirin or γT alone, suppressed colon tumorigenesis in mice, as indicated by 40% and 50% reduction in the multiplicity of total polyps (P < 0.05) and large adenomatous polyps (>2mm2, P < 0.05), respectively. More strikingly, the combination of aspirin and γT reduced the overall tumor area by 60% (P < 0.05). Noteworthy, the supplementation of γT also alleviated aspirin-induced stomach lesion and appeared to modulate intestinal microbial composition. Our study demonstrated that the combination of aspirin and γT has stronger anticancer activity than aspirin or γT alone while alleviates aspirin-associated adverse effect, suggesting that the combination of γT and aspirin is a more effective and safer chemopreventive agent for CRC than aspirin alone.</p>

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A major challenge in designing cancer therapies is the induction of cancer cell apoptosis, although activation of intrinsic apoptotic pathways by targeting gold nanoparticles to mitochondria is promising. We report an in vitro procedure targeting mitochondria with conjugated gold nanoparticles and investigating effects on apoptosis induction in the human breast cancer cell line Jimt-1. Gold nanoparticles were conjugated to a variant of turbo green fluorescent protein (mitoTGFP) harbouring an amino-terminal mitochondrial localization signal. Au nanoparticle conjugates were further complexed with cationic maltotriose-modified poly(propylene imine) third generation dendrimers. Fluorescence and transmission electron microscopy revealed that Au nanoparticle conjugates were directed to mitochondria upon transfection, causing partial rupture of the outer mitochondrial membrane, triggering cell death. The ability to target Au nanoparticles into mitochondria of breast cancer cells and induce apoptosis reveals an alternative application of Au nanoparticles in photothermal therapy of cancer.

IGFBP-3 expressing rekombinant vaccinia viruses used for tumor therapy Insulin-like growth factor-binding protein-3 (IGFBP-3) is a major regulator of endocrine effects of IGF and is capable to suppress the growth of variety of cancer. Several studies have shown that IGFBP-3 can induce the apoptosis of cancer cells via IGF-dependent and IGF-independent mechanisms. In our study, we have constructed recombinant vaccinia viruses (VACV) expressing IGFBP-3 under the control of the early H5 and synthetic early/late (E/L) promoter to investigate the potential effect on cancer growth in our cervical cancer model. We have shown that the expression of IGFBP-3 alone had no effect on tumor growth. On the other hand, the co-expression of IGFBP-3 enhanced the anti-cancer effect of immunization with the fusion protein SigE7LAMP, which gave rise to the anti-cancer immunity directed against HPV16 induced tumors. We have shown that the double-recombinant P13-SigE7LAMP-H5-IGFBP-3 can enhance the protective immune responses against MK16/ABC induced tumors. Furthermore, we have show that both double-recombinant viruses P13-SigE7LAMP-H5- IGFBP-3 and P13-SigE7LAMP-E/L-IGFBP-3 can increase the anti-cancer effect of SigE7LAMP expression in the therapy of TC-1 induced tumors. Key words: IGFBP-3, IGF, VACV, HPV16, E7 oncoprotein,...