Cancer is one of the leading causes of death. There have been many investigations into therapeutic ways to prevent and reverse cancerous growth. We report a new approach in this thesis, which is to investigate the functions of Vitamin B1 (thiamine) in cancerous cells and their regulation. A number of thiamine analogs were synthesized to carry out the structure-activity relationship (SAR) studies with two transporters THTR1 and THTR2. Initial results show that the modifications of thiazole reduced the uptake of thiamine.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most insidious forms of cancer whose incidence nearly equals its death rate. Despite extensive research studies, no effective therapeutic approaches for diminishing the morbidity associated with this disease are available. PDAC is characterized by activating Kras mutations and inactivation of Ink4a and the p53-Arf pathway in virtually all cases, while SMAD4¡Xa central regulator of Transforming growth factor-beta (TGF-£]) signaling¡Xis inactivated in 55% of PDAC. Our overall goal is to understand how perturbations in the inactivation of SMAD4 pathway contribute to the late stages of PDAC pathogenesis, and to elucidate the role of SMAD4 inactivation on the conversion of a benign form of the cancer to a more aggressive metastatic form. To address this important topic in cancer biology, we have devised a strategy to develop model cell lines to dissect the role of SMAD4 defect in PDAC cell lines and the potential synergistic effects of hypoxia and/or TGF-£]1 upon SMAD4 inactivation in their metastatic properties. Experiment results showed SMAD4 restored in PDAC model cell lines were down regulate HIF-1£\, VEGF, FGF10 and FGFR2 genes expression level, and also inhibited migration, chemoresistance and angiogenesis of cancer cells. We hypothesize that these effects are due to SMAD4 suppresses some cancer genes in PDAC. Further detailed investigations are also needed to fully elucidate the detail mechanisms for our findings here therefore, the future works of this study will go step on looking for those important downstream effect genes regulated by Smad4 protein in PDAC cells and try to find out the connection of all the dependence proteins.

Previous studies revealed that, upon exposure to hypoxia, tumour cells acquire resistance to the cytolytic activity of IL-2-activated lymphocytes. The MHC class I chain-related (MIC) molecules – comprised of MICA and MICB – are ligands for the activating NKG2D receptor on Natural Killer (NK) and CD8+ T cells. MIC-NKG2D interactions lead to the activation of NK and CD8+ T cells and the subsequent lysis of the tumour cells. The study also showed that the mechanism of the hypoxia-mediated immune escape involves the shedding of MIC, specifically MICA, from the tumour cell surface. The objective of the present study was to determine whether the shedding of MICA requires the expression of hypoxia inducible factor-1 (HIF-1), a transcription factor that regulates cellular adaptations to hypoxia. Exposure to hypoxia (0.5% O2 vs. 20% O2) led to the shedding of MIC from the surface of MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells as determined by flow cytometry. Knockdown of HIF-1α mRNA using siRNA technology resulted in inhibition of HIF-1α accumulation under hypoxic conditions as determined by Western blot analysis. Parallel study revealed that knockdown of HIF-1α also blocked the shedding of MICA from the surface of MDA-MB-231 cells exposed to hypoxia. These results indicate that HIF-1 is required for the hypoxia-mediated shedding of MICA and, consequently, that HIF-1 may play an important role in tumour immune escape. Ongoing studies aim to determine the HIF-1 target genes involved in the shedding of MICA under hypoxia. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2009-08-19 21:09:13.707

Introduction: Since the era of Marshall, H. pylori has been to be implicated in many upper digestive tract diseases, such as gastritis, peptic ulcer disease, mucosa- associated lymphoid tissue lymphoma, and even gastric adenocarcinoma. In 1994, WHO recognized H. pylori as a definite carcinogen for gastric cancer. Many study had shown that microbial pathogens may induce oxidative stress in infected host cell. And this may also represent an important mechanism leading to epithelial injury in H. pyloric infection. Oxidative stress plays a role in altering epithelial cell turnover, accelerating apoptosis and increasing oxidative DNA damage. One of the evidences for this phenomenon is increasing level of reactive oxygen species (ROS) measured in the mucosa of infected stomach. ROS may activate HIF-11£\ transcription. HIF-1£\ overexpression had been detected in several human cancers. Furthermore its overexpression correlates significantly with highly aggressive disease, lymph node metastasis, clinicopathological status and poor prognosis in some cancer types. It may up regulate hypoxia-induced gene, such as the vascular endothelial growth factor (VEGF) transcription and angiogenesis. Therefore we propose Sp1-pVHL-HIF-1£\ pathway may play a role the carcinogenesis in Hp -associated gastric cancer. Material and methods: We took Paraffin-embedded specimens from 89patients, who had undergone UGI endoscopy and gastric mucosa biopsy. We assessed the Sp1, pVHL, HIF-1£\ in all cases by immunohistochemistry and then evaluated their correlation with the H pylori infection. Chi-square and Fisher¡¦s exact test was performed to determine the significance of the difference between Sp1, pVHL, HIF-1£\. Results: There are not significant difference in nuclear Sp1 expression and H. pylori different (p=0.59). Sp1 expression was not significant, (p=0.91, 0.93, 0.36, 0.42, 0.51) with sex, age, location, TNM stage and cell differentiation. pVHL protein was mainly expressed in the cytoplasm. There are no significant difference with H. pylori infection (p=0.14). The relationship pVHL protein expression between with sex, age, lesion site, TNM stage and cell differentiation were not significant (p=0.39, 0.70, 0.69, 0.83, 0.70). HIF-1£\ protein was mainly expressed in the nuclei. There are not significant association with H. pylori infection (p=0.49). There were no significant differences between HIF-1£\ protein expression with sex, age, location, TNM stage and cell differentiation (p=0.94, 0.32, 0.75, 0.35, 0.60). Furthermore, In normal tissue the expression of HIF-1£\ had significant association with pVHL(p=0.0002), and the expression also had no a mariginally significant association with Sp1(p=0.096). Expression of Sp1 had significant association with pVHL(p=0.0016)in tumor tissue, Therewas a significant association between normal and tumor tissue expression of the pVHL and Sp1(p=0.038, 0.019), but the expression of HIF-1£\ had no significant(p=0.23). Conclusion: In this study, we attempt to determine the association between Sp1-pVHL -HIF-1£\ pathway and a role in the carcinogenesis in H. pylori infection. Although we didn¡¦t confirm the hypothesis Sp1-pVHL -HIF-1£\ pathway playing an critical role in the mechanism of gastric cancer. We concluded that there is no significance between the expression of Sp1, pVHL and HIF-1£\ and gastric cancer, but the role of this pathway in the Hp infection associated carcinogenesis is still to be clarified.

Hypoxia Inducible Factors (HIFs) are very important transcription factors that can respond to low oxygen concentrations in the cellular environment. Inhibition of HIF’s transcriptional activity represents a promising approach to new anticancer compounds. Herein, we describe the design and synthesis of a series of HIF-1 inhibitors. Evaluation of these inhibitors using a cell-based luciferase assay led to the discovery compounds with sub-micromolar potency.

Le cancer est une pathologie complexe causée par plusieurs facteurs génétiques et environnementaux. Aux États-Unis seulement, près de 1 400 000 cas de cancer seront diagnostiqués en 2006 et plus de 500 000 personnes en mourront. Encore aujourd'hui, plusieurs types de cancer sont incurables et la plupart des traitements existants ne sont malheureusement pas curatifs. Cette maladie est caractérisée par une croissance cellulaire incontrôlée ainsi qu'une dissémination des cellules anormales. La prolifération accrue des cellules cancéreuses mène à l'apparition de régions hypoxiques chez plusieurs tumeurs solides. Un grand nombre de molécules participent aux différents stades de la progression tumorale et permettent à la tumeur de s'adapter à son microenvironnement. Plusieurs de ces dernières sont synthétisées sous forme de pro-peptides nécessitant un clivage enzymatique ciblé afin d'acquérir leur activité. Il a été démontré que la furine, le prototype de la famille des convertases de proprotéines, joue un rôle important dans la progression tumorale. La furine est surexprimée chez plusieurs types de tumeurs et elle est nécessaire à l'activation de substrats protéiques dont les rôles dans la croissance tumorale ainsi que dans la formation des métastases sont répertoriés. Dernièrement, l'étude des différents promoteurs de la furine a révélé qu'elle pouvait être induite en hypoxie, et. ce, via le facteur de transcription HIF-1. Notre étude avait pour but d'étudier l'implication de la régulation hypoxique de la furine dans le développement du cancer. Nous avons démontré, par immunohistochimie, la présence de régions hypoxiques dans les xénogreffes formées à partir de fibrosarcomes humains ainsi qu'une surexpression de la furine dans ces régions. Afin d'inhiber cette induction dans les régions hypoxiques tumorales, nous avons construit des vecteurs plasmidiques contenant un inhibiteur de la furine, soit l'[alpha]1-AT-PDX, sous le contrôle d'un promoteur inductible en hypoxie. Ces vecteurs possèdent 3 ou 6 répétitions d'une séquence du gène de l'érythropoïétine contenant un site de reconnaissance pour le HIF-1. Nous avons démontré que ces vecteurs augmentent les niveaux de PDX chez les cellules HT1080 cultivées en hypoxie. De plus, nous avons établi que l'augmentation de PDX en hypoxie est principalement due à la présence de HIF-1 dans la cellule. Nous avons aussi observé une diminution de la maturation de la MT1-MMP ainsi que de l'IGF-IR[bêta], deux substrats de la furine. Dans le but de vérifier la possibilité qu'une inhibition de la furine induite en hypoxie puisse affecter la croissance tumorale, nous avons par la suite transfecté les cellules HT1080 afin d'obtenir une population de cellules exprimant de façon stable les plasmides 3HRE-PDX et 6HRE-PDX. Nous avons démontré que, suite à l'implantation de ces cellules chez des souris immunodéficientes, la croissance des tumeurs exprimant du PDX en hypoxie est diminuée de façon significative. Nous avons également démontré, par immunobuvardage de type Western, la présence de PDX à l'intérieur de ces tumeurs. De plus, des expériences d'immunohistochimie ont révélé que l'expression de PDX était principalement située dans les régions hypoxiques des tumeurs. La furine est une enzyme ubiquiste, impliquée dans le maintien physiologique des fonctions cellulaires. Nos résultats démontrent pour la première fois la faisabilité d'inhiber de façon ciblée la furine, soit dans les zones hypoxiques de tumeurs. Cette inhibition diminue la maturation de substrats impliqués dans la modification du microenvironnemt des tumeurs et par conséquent, diminue la progression tumorale.

Cancer accounts for nearly one-quarter of deaths in the United States, exceeded only by heart diseases. In 2006, there were 559,888 cancer deaths in the US. Finding effective treatments for cancer is a major challenge among researchers. In solid tumor, hypoxia increases the progression of malignancy and metastasis by promoting angiogenesis. The transcription factor HIF-1 is responsible for the regulation of cellular processes, including glycolysis and angiogenesis. Clinical evidence has determined that expression of HIF-1 is strongly associated with poor patient prognosis. Also, activation of HIF-1 contributes to malignant behavior and therapeutic resistance. In view of these observations, there is a need for anti-cancer treatments that addresses hypoxic related tumors. HIF-1 presents a viable target for inhibition of tumor growth with small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 pathway, as well as mechanistic studies involved in the investigation of the mode of action of these compounds.

In most organisms the cellular response to hypoxia is mediated by the master regulator hypoxia-inducible factor-1 (HIF-1). Zebrafish embryos can also arrest development (suspended animation) to tolerate low oxygen. I tested the hypothesis that induction of HIF-1 and associated target genes (eg. erythropoietin) during embryonic development would alter the hypoxia tolerance phenotype of larval and adult fish. I exposed zebrafish embryos at 3 developmental stages to acute (4 h) bouts of hypoxia (5% dissolved oxygen, DO) or anoxia (<0.5% DO). I found that embryos that mount a HIF-1 response have a greater hypoxia tolerance as larvae. Additionally, populations that experienced embryonic HIF-1 induction show an increase in the proportion of males (~70% male), that are more hypoxia tolerant than female fish, compared to control populations (~45% male). Overall, induction of HIF-1 during ontogeny alters the larval and adult zebrafish phenotype to better tolerate future hypoxic bouts. / NSERC

Throughout history, cancer has been severely plaguing mankind; the search for a cure to cancer had long been sought by scientists and still poses as one of the greatest challenges scientists have yet to overcome. Hypoxia in cells is a condition where there is little to no oxygen availability in its environment. In general, this event is detrimental since this can lead to cell necrosis or reoxygenation injuries. However, hypoxia, a prominent property of most solid tumors, activates the hypoxia-inducible factor (HIF-1) family of transcription factors that promotes angiogenesis. In this study, we describe the design and synthesis of small-molecule inhibitors of the HIF-1 pathway.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ceilândia, Pós-Graduação em Ciências e Tecnologias em Saúde, 2016. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2016-09-08T14:50:10Z No. of bitstreams: 1 2016_MarcusAuréliodaCostaTavaresSabino.pdf: 1902510 bytes, checksum: 9746d9bbcc04914de08edc18633f28c0 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-11-08T11:12:37Z (GMT) No. of bitstreams: 1 2016_MarcusAuréliodaCostaTavaresSabino.pdf: 1902510 bytes, checksum: 9746d9bbcc04914de08edc18633f28c0 (MD5) / Made available in DSpace on 2016-11-08T11:12:37Z (GMT). No. of bitstreams: 1 2016_MarcusAuréliodaCostaTavaresSabino.pdf: 1902510 bytes, checksum: 9746d9bbcc04914de08edc18633f28c0 (MD5) / Nos animais sensíveis à privação de O2, é observada a injúria tecidual durante a hipóxia e o reoxigenação, tendo como causa principal o estresse oxidativo gerado por radicais livres derivados do oxigênio. Uma das estratégias encontradas por uma ampla variedade de animais à escassez de oxigênio é responder a este insulto com aumento das defesas antioxidantes, e assim evitar o dano oxidativo causado pelos radicais livres, fenômeno nomeado de preparo para o estresse oxidativo (POS). Nossa pergunta é saber se o fenômeno bioquímico do POS acontece na natureza, em especial, no ambiente entre marés, no qual mudanças cíclicas de retirada e oferta de oxigênio acontecem durante a baixa e alta das marés. Nestas condições de baixa e alta da maré em uma praia de Penha (Santa Catarina, Brasil), foi estudado o metabolismo redox envolvendo glutationa (GSH), em análise de corpo inteiro nos mexilhões da espécie Mytilaster solisianus, em três diferentes coletas (chamadas de I, II e III). Os animais mostraram suportar o esperado desequilíbrio redox durante as situações de exposição aérea (de até quatro horas na coleta I e II, e até 9 horas na coleta III) e reimersão no campo, pois os parâmetros antioxidantes endógenos relacionados ao metabolismo de GSH estiveram inalterados durante o ciclo das marés (i.e. Eq-GSH, GSH, GSSG e GSSG/Eq-GSH). Todavia na coleta III, houve uma queda dos níveis totais de Eq-GSH após duas horas de exposição aérea em relação ao grupo de animais pré-imersos. Na coleta III houve uma forte variação da temperatura do ar ao longo da exposição aérea (de 19 para 28° C), apesar disso, o indicador de desequilibrio redox não mudou (razão GSSG/Eq-GSH), mostrando que estes animais têm alto grau de adaptabilidade neste ambiente, no qual os fatores abióticos estão em constante mudança. _________________________________________________________________________________________________ ABSTRACT / In animals sensible to oxygen privation is seen injury in their tissues during hypoxia and reoxygenation, and the main cause is the oxidative stress generated by oxygen-derived free radicals. One of the strategies found by a widespread variety of animals to survive oxygen lack is respond with increase of antioxidant defenses, and thus avoiding the oxidative damage caused by free radicals, a phenomenon named preparation for oxidative stress (POS). Our question address if biochemical phenomenon of POS happens in nature, in special, in the intertidal environment, where changes cyclical of lack and supply oxygen happens during low and high tide. In these conditions of low and high tide in beach of Penha (Santa Catarina, Brazil), was studied redox metabolism of glutathione (GSH), in body whole analysis in the mussel specie Mytilaster solisianus in three differents expeditions (named of I, II e III). The animals shown endure to the expected redox imbalance during aerial exposure (until four hours in expedition I e II, and until 9 hours in expedition III) and reimmersion in field, because the endogenous antioxidant parameters related to the metabolism of the glutathione remained unchanged during tidal cycle (i.e GSH-eq, GSH, GSSG and GSSG/GSH-eq ratio). However in expedition III, there was a drop in glutathione pool (GSH-eq) after two hours of aerial exposure when compared to group of animals pre-emersion. In expedition III there was a strength variation in air temperature (19 to 28° C) during aerial exposure, despite this, the index of redox imbalance remained unchanged (GSSG/GSH-eq) in expedition III, showing that mussels have high level of adaptability in this environment, in which abiotic factors are in continuous changes.