no / The cytochrome P450 family of enzymes is involved in the Phase I metabolism of a wide variety of compounds. Although generally involved with detoxification, overexpression of one family member, cytochrome P450 1B1 (CYP1B1), has been associated with human epithelial tumors. As such, CYP1B1 was hypothesized to be a novel target for the development of anticancer therapies. We investigated expression of CYP1B1 protein in 61 human colorectal adenocarcinomas and compared this to that observed in 14 histologically normal human large bowel samples removed from patients undergoing surgery for large bowel tumors. Although we confirmed that CYP1B1 was expressed at high levels in human colorectal tumor epithelia, we also found that CYP1B1 was not absent from normal colonic epithelia but was expressed at low levels. The expression of CYP1B1 in colon tumors does not correlate with tumor stage or degree of lymph node invasion in this study. Furthermore, in addition to expression in colon epithelia, CYP1B1 is also observed in blood vessels within the colon. As with the epithelia, levels of CYP1B1 were higher in tumor vasculature than that of the normal colon. Although these observations greatly support the development of CYP1B1 targeted anticancer therapies, they also indicate the caution that should be observed when developing such drugs.

In the interest of identifying natural substances from marine invertebrates collected off the waters of Taiwan, we have searched the bioactive metabolites from the organic extracts of gorgonian corals Rumphella antipathies and Echinomuricea sp. This study had led to the isolation of thirty compounds (1¡V30), including nine new caryophyllane-related metabolites, rumphellaones A (1), B (2) and C (3), rumphelloic acids A (4) B (5) and C (6), rumphellolides J (7), K (8) and L (9), five new clovane-related metabolites, rumphellclovanes A (12), B (13), C (14), D (15) and E (16), two new disesquiterpenoid dimers, rumphelladimers A (24) and B (25), eight new natural products, (8R,9R)-isocaryolane-8,9-diol (10), 4£],8£]-epoxycaryophyllan-5-ol (11), 9£\-hydroxyclovan-2-one (17), 2£]-hydroxyclovan-9-one (18), clovan-2,9-dione (19), 2£]-acetoxyclovan-9£\-ol (20), 9£\-acetoxyclovan-2£]-ol (21) and 2£],9£]-dihydroxyclovane (22), along with a known compound, clovan-2£],9£\-diol (23) from Rumphella antipathies. In addition, three new labdane-, halimane-, and clerodane-related metabolites, echinolids A (26), B (27) and C (28), a new sesquiterpenoid natural product, (7S,10R)-(+)-10,11-epoxycurcuphenol (29), along with a known compound, (+)-curcuphenol (30) were also found in Echinomuricea sp. The structures of metabolites 1¡V30 were established by spectroscopic methods and by comparison of the spectral data with those of related known compounds. The absolute configurations of clovane-type compounds were determined using a modified Mosher¡¦s method for 23. The biosyntheses of compounds 1¡V5 and 12 were proposed. In the biological activity experiments, compounds 5 and 19 displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB. Compounds 24 and 27 displayed significant inhibitory effects on elastase release by human neutrophils. Compound 27 was found to exhibit inhibition against the growth of DLD-1 (human colon adenocarcinoma) and Lovo (human colorectal adenocarcinoma) tumor cells.

No / Despite promising early data, the natural product dolastatin 10 has not been successful as a single agent in phase II clinical trials. Herein the mechanism of action and efficacy of a synthetic analogue, auristatin PYE, was investigated in 2 human colon adenocarcinoma models, DLD-1 and COLO 205. In vivo efficacy was assessed in subcutaneous xenografts following intravenous administration. Mechanistic studies investigated effects of auristatin PYE on microtubule disruption using immunocytochemistry, whilst cell cycle effects were studied using flow cytometry. Possible effects on tumour functional blood vasculature were assessed in tumour-bearing mice. Auristatin PYE was less potent in vitro than dolastatin 10, but was significantly more effective (p<0.01) in vivo against both tumours. Significant effects on tumour blood vasculature were seen, with optimal shutdown at 6-h post-treatment. Extensive necrosis became more evident over time after treatment. Auristatin PYE caused severe disruption of normal microtubule structure at concentrations and times comparable with the IC50 data, and also instigated a G2/M cell cycle block. Auristatin PYE was more effective in the DLD-1 and COLO 205 models than dolastatin 10, with anti-tumour effects mediated through vascular shutdown. These data suggest that auristatin PYE has good potential as an anti-cancer agent.

Introduction: Functional motility disorders of the colon are poorly defined. Hence, patients with chronic colonic motor dysfunction are treated or undergo surgery without proper diagnosis. Most colonic motility assessment centers around the largest propagating motor pattern in the colon- the High Amplitude Propagating Pressure Wave (HAPW). However, there is no consensus regarding a definition of this important motor pattern. Additionally, no consideration is given to other aspects of colonic motility such as colo-ano-rectal coordination and control by the autonomic nervous system (ANS). The aim of this thesis was to improve understanding of HAPWs and other features of colonic motility in health and constipation, understand how autonomic dysfunction is related to observations in patients, and evaluate the effect of neuromodulation of the ANS. Methods: Motility was assessed in healthy volunteers and patients using water-perfused High Resolution Colonic Manometry (HRCM). To assess the association between ANS and colonic motor activity Heart Rate Variability (HRV) was measured in patients. Spatiotemporal maps were created using HRCM to analyse and quantify colonic motor activity following baseline, and interventions which included proximal balloon distention, meal, and rectal bisacodyl. Low-Level Light Therapy (LLLT) was also applied during HRCM as a method of neuromodulation, to observe its effect on colonic motility. Results: Normal HAPWs are those which have an amplitude of more than 50 mmHg and belong to one of 3 categories: proximally originating, proximal continuing, and transverse/descending. The best intervention sequence to generate these during HRCM assessment is baseline, proximal balloon distention, meal, rectal bisacodyl. Based on their responses to these interventions and the type of HAPWs present, patients could be classified into strong responder, weak responder and non-responder groups. Overall, patients in the strong responder group were most similar to healthy volunteers both with regard to motility and ANS control. Conversely, the weak and non-responders had showed decreased or no motility with decreased parasympathetic input and occasionally sympathetic inhibition. Additionally, other features of motility such as the sphincter of O'Beirne, and lack of colo-ano-rectal coordination were found to lead to constipation even in presence of normal HAPWs. LLLT shows promise in initiating colonic motor activity through neuromodulation of the sacral defecation center. Conclusions: HAPWs can be defined into one of three categories and used to categorize patients based on their HAPW response to different interventions. However, other aspects of colonic motility such as the colo-ano-rectal coordination and autonomic nervous system control of colonic motility should be taken into consideration in diagnosis of constipation, as they can point towards more non-invasive treatment methods such as neuromodulation using LLLT. / Thesis / Master of Science in Medical Sciences (MSMS)

Le sulfure d'hydrogène (H2S) est un métabolite bactérien produit notamment par les bactéries sulfato-réductrices du côlon à partir des acides aminés soufrés, des sulfates/sulfites alimentaires et des sulfomucines. A fortes concentrations, le H2S est un gaz toxique, de par sa capacité à inhiber la cytochome c oxydase, et par conséquent, la respiration mitochondriale. A l'inverse, notre travail montre qu'à faibles concentrations, le H2S induit une énergisation mitochondriale des cellules épithéliales coliques humaines HT 29 Glc-/+ lui conférant aussi un rôle de substrat minéral énergétique. Dans ce travail, nous avons déterminé les concentrations de H2S permettant l'oxydation/détoxication de H2S par les cellules HT 29 Glc-/+ et celles provoquant une inhibition de la consommation d'oxygène. L'oxydation du H2S nécessite la coopération entre la « sulfide oxidation unit » et la chaîne respiratoire. La capacité des cellules HT29 Glc-/+ à oxyder le H2S est associée à la présence des transcrits codant les enzymes constituant la « sulfide oxidation unit » : la sulfure d'hydrogène quinone reductase (SQR), la dioxygenase ETHE1 et la thiosulfate transferase (TST). Nous avons démontré la priorité de l'oxydation du H2S sur les substrats carbonés . En effet, nos résultats suggèrent que les électrons venant de la SQR sont transférés au pool d'ubiquinone aux dépens de ceux venant du complexe I. Nos résultats démontrent que la SQR joue un rôle déterminant pour l'oxydation de H2S. De plus, la détoxication du H2S par les cellules HT29 Glc-/+ augmente au cours de la différenciation spontanée ou induite par un traitement au butyrate. L'augmentation de la détoxication de H2S au cours de la différenciation est associée à une augmentation de la réserve respiratoire soulignant l'importance de la chaîne respiratoire comme composante de la fonction de détoxication du H2S. En situation d'inhibition de la cytochrome c oxydase, la grande capacité des cellules coliques humaines à détoxiquer le H2S pourrait être en partie due à la présence d'un transfert réverse des électrons issus de l'oxydation de H2S de la SQR vers le complexe I. Outre le butyrate, le zinc un autre composé de la lumière colique, exerce un effet protecteur contre la toxicité cellulaire du H2S. Enfin, notre travail a mis en évidence une diminution de l'expression d'un gène codant pour une enzyme de la « sulfide oxidation unit » (la TST) dans le rectum comparée à différents segments du côlon, ce qui pourrait correspondre à des capacités de détoxication du H2S différente en fonctions des segments du gros intestin humain. / Hydrogen sulfide (H2S) is a metabolite produced notably by colonic sulphate-reducing bacteria from alimentary sulphur-containing amino acids, sulfates / sulfites and sulfomucines. At high concentrations, H2S is a toxic gas due to its ability to inhibit cytochome c oxidase, and therefore mitochondrial respiration. In contrast, our study shows that at low concentrations, H2S induces mitochondrial energization in human colonic epithelial cells HT-29 Glc -/+ assigning it a role as the first inorganic oxidative substrate in human cells. In this work, we have determined sulphide concentrations allowing sulphide oxidation/detoxification by HT-29 cells and those which inhibit oxygen consumption. The oxidation of H2S requires cooperation between the “sulphide oxidation unit” and the respiratory chain. The capacity of HT-29 Glc -/+ to oxidize H2S is associated with the presence of transcripts encoding the enzymes constituting the “sulfide oxidation unit”: the sulphide quinine reductase (SQR), the sulphur dioxygenase or Ethylmalonic encephalopathy 1 (ETHE1) and the thiosulfate sulphur transferase (TST). We demonstrate that the oxidation of H2S takes precedence over the oxidation of carbon substrates. Indeed, our results suggest that electrons from SQR are transferred to the ubiquinone pool at the expense of those originating from the complex I. Our results point out that SQR represents a determinanat factor in the oxidation of H2S. In addition, the detoxification of H2S by HT-29 Glc -/+ cells increases during spontaneous differentiation and differentiation induced by treatment with butyrate. The increase in the detoxification of H2S during the differentiation is associated with an increase of the respiratory reserve pointing out the importance of the respiratory chain as a component of the detoxification function of H2S. In situations of inhibition of cytochrome c oxidase, the capacity of human colon cells to detoxify H2S could be due in part to the presence of a reverse transfer of electrons from the oxidation of H2S to the SQR complex I. In addition to butyrate, zinc, another compound of the colonic lumen has a protective effect against cell toxicity associated with H2S. Lastly, we have shown a decreased expression of the gene encoding an enzyme of the “sulfide oxidation unit” (TST) in the rectum compared to the other segments of human colon. This observation may correspond to different detoxification capacities towards H2S according to the different parts of the human large intestine.

Made available in DSpace on 2014-08-20T13:32:46Z (GMT). No. of bitstreams: 1 tese_fernanda_nedel.pdf: 5232153 bytes, checksum: 94c9f4584571a1e142e44a8b165fc8dd (MD5) Previous issue date: 2012-09-17 / Cancer is a leading cause of death and its rates are expected to increase 50% by 2020. Although surgical resection and additional therapies (such as chemotherapy and radiotherapy) are able to cure well-confined, primary tumors, the same does not apply during metastasis due to the systemic involvement and its resistance to conventional therapies. Therefore, the current clinical challenge is to develop new drugs and treatment modalities that will significantly impact the cure rates. In this sense, the present study aimed to evaluate the anticancer effect and study the underlying cell death mechanisms of diaryl diselenides and its substituted structures - (4-ClC6H4Se)2, (3-CF3C6H4Se)2 e (4-MeOC6H4Se)2 - on the human colon adenocarcinoma cell line (HT-29). We verified that (3-CF3C6H4Se)2 and (4-MeOC6H4Se)2 induced cytotoxicity through apoptosis mechanisms in HT-29 cells, where pro-apoptotic genes were up-regulated (Bax, caspase-9, caspase-8, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), and anti-apoptotic genes were down-regulated (Bcl-2 and survivin). In a second moment we evaluated the anticancer potential of Canavalia brasiliensis (ConBr), Canavalia boliviana (ConBol) and Canavalia ensiformis (ConA) lectins in HT-29 cells, which showed an effective capacity to reduce cell viability. Once the anticancer effect was confirmed, lectins were labeled with FITC and its interaction with the tumor cells was investigated. The FITC-ConA and FITC-ConBol demonstrated the potential to bind to HT-29 cells unlike FITC-ConBr. In order to investigate a new treatment modality, the interaction between the respective lectins with HT-29 was evaluated when associated with functionalized multi-walled carbon nanotubes (f-MWCNTs). When f-MWNT was incorporated to FITC-ConBol and FITC-ConA lectins there was an increase in fluorescence intensity. / O câncer é uma das principais causas de morte no mundo, onde os índices devem aumentar 50% até 2020. Embora a ressecção cirúrgica e terapias adicionais (como a quimioterapias e radioterapias) sejam capazes de curar tumores primários bem delimitados, o mesmo não se aplica a metástase devido ao seu envolvimento sistêmico e a resistência a terapias convencionais. Portanto, atualmente o desfio clínico é desenvolver novas drogas e modalidades de tratamentos que irão impactar significativamente as taxas de cura do câncer. Neste sentido, o presente trabalho objetivou avaliar o efeito antineoplásico e investigar a rota de apoptose induzido pelo disseleneto de diarila e seus derivados substituídos - (4-ClC6H4Se)2, (3-CF3C6H4Se)2 e (4-MeOC6H4Se)2 - em células de adenocarcinoma de colorretal humano (HT-29). Verificamos que os compostos (3-CF3C6H4Se)2 e (4-MeOC6H4Se)2 induziram um efeito citotoxidade por meio de apoptose, onde os genes pró-apoptoticos (Bax, caspase-9, caspase-8, fator indutor de apoptose (AIF) e endonuclease G (EndoG)) foram altamente expressos e os genes anti-apoptótico (Bcl-2 e survivin) mostraram uma redução na sua expressão. Em um segundo momento avaliamos o potencial antineoplásico das lectinas Canavalia brasiliensis (ConBr), Canavalia boliviana (ConBol) e Canavalia ensiformis (ConA) em células HT-29, as quais se mostraram efetivas em reduzir a viabilidade celular. Uma vez confirmado o efeito antineoplásico, as lectinas forma marcadas com FITC e a sua interação com as células tumorais foi investigado. As lectinas FITC-ConA e FITC-ConBol demonstraram potencial de se ligar as células HT-29 ao contrário da FITC-ConBr. A fim de investigar uma nova modalidade de tratamento foi avaliada a interação entre as respectivas lectinas com as células HT-29 quando associadas à nanotubos de carbonos funcionalizados de paredes múltiplas (f-MWCNTs). Quando os f-MWCNTs foram incorporados as lectinas FITC-ConA e FITC-ConBol houve um aumentaram na intensidade de fluorescência.