We have hypothesized that dietary modulation of intestinal miRNA expression may contribute to the chemoprotective effects of nutritional bioactives (fish oil and pectin). Using a rat colon carcinogen model, we determined miRNAs-let-7d, miR-15b, miR-107, miR-191 and miR-324-5p were modulated by fish oil + pectin. We also demonstrated that BACE1 and PTEN are targets of miR-107 and miR-21, respectively. To further elucidate the biological effects of diet and carcinogen on miRNAs, we integrated global miRNAs, total and polysomal gene expression datasets obtained from the above mentioned study and used four computational approaches. We demonstrated that polysomal profiling is tightly related to microRNA changes when compared with total mRNA profiling. In addition, diet and carcinogen exposure modulated a number of microRNAs and complementary gene expression analyses showed that oncogenic PTK2B, PDE4B, and TCF4 were suppressed by the chemoprotective diet at both the mRNA and protein levels. To determine the function of select diet and colon carcinogen modulated miRNAs and to validate their targets, we carried out a series of loss and gain of function experiments along with luciferase reporter assays. We verified that PDE4B and TCF4 are direct targets of miR-26b and miR-203, respectively. PTK2B was determined to be an indirect target of miR-19b. In addition, microRNA physiological function was assessed by examining effects on apoptosis and cell proliferation. To better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we investigated the chemoprotective effects of dietary agents on miRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creERT2 knock in mice injected with AOM. We demonstrated that based on relative expression of miR-125a-5p, miR-190b and miR-191 in stem cells vs. daughter cells and differentiated cells, these miRNAs may be stem cell specific miRNAs. We also identified miR-21 to be significantly reduced in stem cells compared to differentiated cells and selectively modulated by these dietary agents in stem cells. In summary, our results indicate for the first time that fish oil plus pectin protect against colon tumorigenesis in part by modulating a subset of miRNAs and their target genes (mRNAs) implicated in the regulation of the colon stem cell niche and tumor development.

Overwhelming evidence indicates that aspirin (ASA) and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity against colorectal cancer (CRC). Although the underlying mechanisms have yet to be fully elucidated, the host laboratory have shown that nucleolar sequestration of the NF-κB component RelA is critical. In the course of these studies, it was noted that alongside effects on the NF- κB pathway, ASA has a profound effect on nucleoli, including a dramatic increase in nucleolar size. These data were particularly interesting as, in addition to its role in ribosome biogenesis, the nucleolus is known to act as a stress sensor and play a key role in the regulation of cell growth and apoptosis. Indeed, this organelle has been identified as a potential target for anti-tumour agents. However, how stress causes changes to nucleolar function, and how these are translated into changes in cell phenotype, remain unclear. Therefore, the aim of my thesis was to fully characterise ASA effects on nucleoli and to determine whether these effects contribute to the anti-tumour activity of this agent. I found that ASA induced an atypical form of nucleolar stress that was associated with enlargement of the organelle, relocalisation of nucleolar markers to the periphery, depletion of the critical component of the Pol I transcription factor complex, TIF-IA, and inhibition of rRNA transcription. These effects were independent of the p38 and JNK2 MAP kinase pathways. However, they were mimicked by inhibition of CDK4, which had previously been shown to lie upstream of ASA effects on the NF-κB pathway. These data describe a novel mechanism by which ASA, and CDK4 inhibition, may inhibit the growth of colon cancer cells. In addition to this candidate approach, I used Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based quantitative proteomics to obtain a global overview of ASA effects on nucleoli of colon cancer cells. Firstly, a protocol was successfully developed to isolate pure nucleoli from SW480 CRC cell lines. This protocol was then applied to SILAC labelled cells treated with ASA for three time-points (0, 6, 10 h). In collaboration with R.T Hay and M. Tatham (University of Dundee), proteomic analysis was then carried out by tandem-mass spectrometry. These data confirmed that ASA has a significant effect on the nucleolar proteome. They also revealed that ASA induces a distinct type of nucleolar stress that is associated with the accumulation of chaperones, translational regulators and members of the ubiquitin-proteasome system (UPS) in this organelle. These data were reminiscent of studies previously published on the effect of proteasome inhibition on nucleoli. I therefore used SILAC-based proteomics to compare ASA effects on nucleoli to those induced by the proteasome inhibitor, MG132. I found that similar sub-groups of proteins accumulate in nucleoli in response to both agents and that ASA induced proteotoxic stress in a similar manner to MG132. Fluorescence correlation spectroscopy in collaboration with R. Duncan and K. Martin (Heriot-Watt University) demonstrated the relative reduction in mobility of nucleolar DsRed-RelA, indicating that, similar to MG132, ASA induces formation of nucleolar aggresomes. Mechanistic studies suggested that blocking ASA-mediated proteotoxic stress blocked the apoptotic effects of the agent. Taken together, these data define a distinct type of nucleolar stress that may be involved in the cells response to proteotoxic stress and be required for the anti-tumour activity of ASA.

Graduation date: 1994

Epidemiological evidence has correlated consumption of sorghum with reduced incidences of gastrointestinal (GI) tract cancer, especially esophageal cancer. There is little evidence on how phenols of sorghum may affect chemoprevention. Seventeen sorghum varieties were screened for phenolic profiles and antioxidant capacity. The ability of crude sorghum extracts to induce NAD(P)H:quinone oxidoreductase (QR, a phase II protective enzyme), and inhibit proliferation of colon (HT-29) and esophageal (OE33) carcinoma cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) and PicoGreen assays, were determined in vitro. 3- Deoxyanthocyanidins, apigeninidin, luteolinidin and their methoxylated derivatives were also investigated for antioxidant capacity, QR inducing and antiproliferative potential. Tannin sorghum generally showed higher antioxidant capacity than non-tannin sorghum varieties. Sorghum varieties containing extractable condensed tannins did not show any significant QR inducing potential; on the other hand, non-tannin sorghums increased QR activity by 1.5-2.7 times; black sorghum (Tx430) was most potent (doubled QR activity at 25 mg/mL, 2.7-fold increase at 100 mg/mL). All sorghum extracts showed relatively strong antiproliferation activity with IC50s (the concentration needed to inhibit cancer cell growth by 50%) of 49.7-883 mg/mL. Tannin-containing sorghums had stronger cancer cell proliferation inhibitory potential (IC50s 49.7-131 mg/mL) than non-tannin sorghums (IC50s 141-883 mg/mL). Total phenol content and antioxidant capacity of crude sorghum extracts positively correlated with their antiproliferative potential (r2 0.71-0.97). Among tested 3-deoxyanthocyanidins, methoxylation on A-ring improved QR inducing potency. 5,7-Dimethoxyluteolinidin had the greatest QR inducing potency (4.3- fold at 100 mM). Methoxylation also improved their antiproliferation potential; the IC50s trend was di-methoxylated (8.3-105 mM) > mono-methoxylated (40.1-192 mM) > apigeninidin and luteolinidin (81.5-284 mM). This study provides information for how phenolic compositions of sorghum and 3-deoxyanthocyanidin structure affect their capacity to induce QR activity and inhibit GI tract cancer cell proliferation. The information is useful to promote the utilization of sorghum in functional foods, beverages, dietary supplements, and other health-related industries. Further study will focus on, fractioned and isolated sorghum phenols, the effect of food processing on their chemopreventive potential, as well as cellular mechanisms involved.

This dissertation investigates the role of limonoids in inflammation to reduce risk of breast cancer and cardiovascular disease. Radical scavenging activity and apoptotic effects of extracts from lemon seeds were investigated in human breast adenocarcinoma (MCF-7) cells and non-malignant breast (MCF-12F) cells. The MeOH:water (80:20) extract showed the highest (29.1%, P < 0.01) inhibition of MCF-7 cells without affecting the non-malignant breast cells. Further, the purified and modified limonoids were screened for their cytotoxicity on estrogen receptor (ER)-positive (MCF-7) or ER-negative (MDA-MB-231) human breast cancer cells. The MCF-7 cell was more susceptible to tested limonoids. Although most of limonoids induced anti-aromatase activity, the inhibition of proliferation was not related to the anti-aromatase activity. On the other hand, the anti-proliferative activity was significantly correlated with the level of caspase-7 activation by limonoids. The next study investigated the mechanism of anti-breast cancer and anti-aromatase activities of obacunone through inhibition of MCF-7 cell proliferation without affecting non-malignant breast cells. Treatment with obacunone resulted in an increased G1 cell cycle arrest and induction of apoptosis. Exposure of MCF-7 breast cancer cells to obacunone down-regulated expression of inflammatory molecules including nuclear factor-kappa B (NF--2 (COX-2). Furthermore, potential of obacunone on inhibition of COX-2 and NF-the p38 mitogen-activated protein (MAP) kinase was also investigated. In the final study, nomilin was the most potent natural inhibitor for p38 MAP kinase activity in human aortic smooth muscle cells indicating that a seven-membered A ring with acetoxy group, present in nomilin, seems to be essential for its inhibitory activity on p38 MAP kinase. The possible mechanism of nomilin for prevention of cardiovascular disease was determined. Pre-treatment with nomilin resulted in significant inhibition of TNF- induced HASMCs proliferation. The anti-proliferative activity of nomilin is due to apoptosis through mitochondrial dependent pathway.

Cisplatin is a commonly used chemotherapeutic agent for multiple solid tumors. However, cisplatin-induced neurotoxicity, nephrotoxicity and hearing loss hamper its use in clinical setting. Although, neurotoxicity and nephrotoxicity can be prevented, there is no cure for cisplatin-induced hearing loss. Cisplatin-induced hearing loss results from damage to outer hair cells (OHCs) in basal turn of the cochlea, to spiral ganglion neurons (SGN), stria vascularis (SV) and fibrocytes of spiral ligament (SL). At the cellular level, cisplatin produces profound increases in reactive oxygen species (ROS) that stimulate cell signaling pathways leading to cochlear inflammation, apoptosis and permanent hearing loss. Thus, potential otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatin chemotherapeutic efficacy. In this study, I characterized the otoprotective actions of the green tea extract, epigallocatechin 3-gallate (EGCG), which possesses anti-oxidant, anti-inflammatory and anti-tumor properties. Oral administration of EGCG to male Wistar rats reduced cisplatin-induced hearing loss, assessed by auditory brainstem responses. These changes were associated with a reduction in cisplatin-induced loss of OHCs primarily in the basal region of the cochlea, along with reduced oxidative stress, inflammatory and apoptotic markers. In addition, EGCG protected against cisplatin-induced decrease in inner hair cell (IHCs) ribbon synapses, labeled with CtBP2. EGCG also protected against cisplatin-induced loss of Na+/K+ ATPase α1 immunoreactivity in the stria vascularis and spiral ligament. In vitro studies using University Bristol/Organ of Corti-1 (UB/OC-1) cells showed that EGCG reduced cisplatin-induced ROS generation and the activation of ERK and STAT1, while it preserved the activity of STAT3 and levels of Bcl-xL. Moreover, EGCG suppressed oxidative stress, inflammatory and apoptotic markers in cisplatin-treated UB/OC-1 cells. Co-administration of EGCG did not alter cisplatin-induced apoptosis of human-derived head and neck cancer cells, ovarian cancer cells or colon cancer cells. In studies using a xenograft model of head and neck cancer in severe combined immunodeficient (SCID) mice, I showed that EGCG did not interfere with cisplatin chemotherapeutic efficacy. These data suggest that EGCG is a potential otoprotective agent for treating cisplatin-induced hearing loss without compromising its chemotherapeutic efficacy.

Made available in DSpace on 2015-07-22T20:50:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-25 / A tuberculose é uma doença infecto-contagiosa, causada pelo Mycobacterium tuberculosis, no Brasil a tuberculose constitui-se em um grave problema de saúde pública, apesar de ser o primeiro país no mundo a implantar o tratamento de curta duração, com multidrogas que garantem a cura em seis meses. A quimioprofilaxia é uma forma preventiva de controle da tuberculose, onde indivíduos sadios, porém infectados pelo bacilo da tuberculose e suscetíveis ao adoecimento, são submetidos ao tratamento medicamentoso por seis meses, conforme preconizado pelo Ministério da Saúde. Este estudo teve como objetivo central avaliar, por meio de um questionário estruturado, as indicações e adesão adequadas da quimioprofilaxia, como prevenção em contatos de pacientes com tuberculose, no município de Palmas – TO. Foram incluídos na pesquisa todos os contatos de doentes de tuberculose, notificados no SINAN, nos anos de 2007 e 2008. Os resultados encontrados revelam que o município de Palmas- TO, através das unidades de saúde, esta atento ao encaminhamento e avaliação médica dos contatos; a adesão ao diagnóstico, ou seja, a primeira consulta enquanto comunicante é de 100%; a prescrição da quimioprofilaxia enquanto prevenção da doença tuberculose é realizada de forma continua, porém a adesão dos pacientes ao tratamento preventivo ainda deixa a desejar, o que é uma preocupante em se tratando de: - Risco de resistência bacteriana, em detrimento da utilização de uma droga; - Risco de adoecer por tuberculose posteriormente; - Risco de perpetuação da cadeia de transmissão da doença; - Aumento de gastos do setor saúde; - Descontinuidade das ações de controle da tuberculose e; - Quebra de vínculo/confiança entre usuário e serviço de saúde. O tratamento preventivo da tuberculose, ou quimioprofilaxia, ocupa importância no que diz respeito à proteção individual de pessoas vulneráveis à tuberculose-doença e ainda um valor social uma vez que o individuo não adoece e conseqüentemente não transmite a doença, quebrando assim a cadeia de transmissão e conquistando a tão sonhada redução de incidência de tuberculose / The tuberculosis is a infectum-contagious illness, caused by the Mycobacterium tuberculosis, in Brazil the tuberculosis consists in a serious problem of public health, although to be the first country in the world to implant the treatment of short duration, with multidrugs that guarantee the cure in six months. The chemoprevention is a preventive form of control of the tuberculosis, where individuals healthy, however infectious for the susceptible bacillus of the tuberculosis and to be sick, are submitted to the drug treatment for six months, as praised for the Health department. This study it had as objective central office to evaluate, by means of a structuralized questionnaire, the adequate indications and adhesion of the chemoprevention, as prevention in contacts of patients with tuberculosis, in the city of Palms - YOU. All Had been enclosed in the research the contacts of tuberculosis sick people, notifying in the SINAN, the years of 2007 and 2008. The joined results disclose that the city of Palmas YOU, through the units of health, this intent to the guiding and medical evaluation of the contacts; the adhesion to the diagnosis, that is, the first contacts consultation while is of 100%; the lapsing of the chemoprevention while prevention of the illness tuberculosis is carried through of form continues, however the adhesion of the patients to the preventive treatment still leaves to desire, what it is a preoccupying one in if treating to: - Risk of bacterial resistance, in detriment of the use of one drugs; - Risk of to be sick later for tuberculosis; - Risk of perpetuation of the chain of transmission of the illness; - Increase of expenses of the sector health; - Discontinuity of the actions of control of tuberculosis e; - Bond In addition/confidence between user and service of health. The preventive treatment of the tuberculosis, or quimioprofilaxia, occupies importance in what it still says respect to the individual protection of vulnerable people to the tuberculosis-illness and a social value a time that the people to be sick and consequently does not transmit the illness, thus breaking the chain of transmission and conquering so dreamed reduction of tuberculosis incidence. / TEDE / BV UNIFESP: Teses e dissertações

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A incidÃncia e mortalidade por cÃncer colorretal apresentam tendÃncia ao crescimento. O cÃncer colorretal à a quinta neoplasia mais incidente no Brasil. A etiologia està relacionada com hereditariedade e modificaÃÃes no estilo de vida. A lesÃo prÃ-neoplÃsica mais precoce com presenÃa de displasia à o foco de cripta aberrante, estando relacionada como lesÃo precursora de adenomas colorretais e cÃncer em humanos. Entender a natureza destas lesÃes contribui na pesquisa de agentes preventivos eficazes no cÃncer colorretal. O objetivo foi verificar o potencial quimiopreventivo da soluÃÃo aquosa do lÃtex de Euphorbia tirucalli L. quanto a formaÃÃo de focos de cripta aberrante (FCA) em ratos induzidos com azoximetano (AOM). Foram usados 32 ratos da linhagem Wistar, machos, com peso mÃdio estimado de 100g -200g (4 â 6 semanas). Foram distribuÃdos aleatoriamente em 04 grupos contendo 08 animais, denominados: GRUPO 01- grupo estudo com ratos induzidos com AOM e tratados com extrato aquoso do lÃtex da E. tirucalli L..GRUPO 02 - grupo estudo controle com ratos induzidos com AOM sem tratamento com extrato aquoso do lÃtex da E tirucalli L..GRUPO 03 - grupo estudo controle sem induÃÃo com AOM e tratados com extrato aquoso do lÃtex da E. tirucalli L.. GRUPO 04 - grupo estudo controle sem induÃÃo com AOM e sem tratamento com extrato aquoso do lÃtex da E. tirucalli L..Os animais dos Grupos 01 e 02 receberam injeÃÃo de AOM 12 mg/kg, intraperitoneal (IP), uma vez por semana, por 02 semanas. Uma semana antes do inÃcio da administraÃÃo do carcinÃgeno, foi administrada diariamente soluÃÃo por gavagem, respectivamente, de extrato aquoso do lÃtex da E. tirucalli L. 400mg/Kg e soluÃÃo fisiolÃgica a 0,9% em uma administraÃÃo diÃria. Os Grupos 03 e 04 nÃo foram induzidos com AOM e receberam soluÃÃo por gavagem, respectivamente, de extrato aquoso do lÃtex da E. tirucalli L. 400mg/Kg e soluÃÃo fisiolÃgica a 0,9%. Todos os grupos continuaram recebendo a soluÃÃo por gavagem diÃria atà o dia estabelecido para eutanÃsia. Foram mortos na 15 semana, apÃs a induÃÃo com carcinÃgeno ou administraÃÃo IP de soluÃÃo estÃril para injeÃÃo. Os animais foram avaliados quanto ao peso, alteraÃÃo clÃnica, presenÃa de adenomas ou tumores cÃlicos, e quanto à presenÃa de FCA e o nÃmero de criptas por cada foco (multiplicidade) de acordo com a localizaÃÃo cÃlica, definidas regiÃo distal, medial e proximal. Verificamos no presente estudo que o extrato aquoso da E tirucalli L. (400mg/kg) apresentou uma diminuiÃÃo significante do nÃmero total de FCA do grupo 01 em relaÃÃo ao grupo 02 (p<0,001), adicionalmente a multiplicidade nestes grupos apresentou diferenÃa significante (p<0,0001) quanto a presenÃa de &#8804; 5 criptas por foco em todo cÃlon examinado. Este estudo sugere que extrato aquoso de E. tirucalli L tem potencial quimiopreventivo em relaÃÃo a carcinogÃnese cÃlica, inibindo a formaÃÃo de FCA em ratos induzidos com AOM. / Colorectal cancer is the fifth most frequently diagnosed malignancy in Brazil. The etiology may be related with inherited and life-style that can be modified. Aberrant crypt foci have been recognized as early preneoplastic lesions and being related with colorectal adenoma and precursors of cancer in humans. Undertanding the nature of early appearing lesions efforts to find effective agents in colorrectal cancer. The aim of this study was verify the potential chemopreventive of aqueous solution of the latex of Euphorbia tirucalli L. in aberrant crypt foci (ACF) in rats induced with azoxymethane (AOM). Thirty-two Wistar male rats were used, average weight 100g -200g (4 - 6 weeks). They were randomly divided into 04 groups of 08 animals each. Group 01 with rats induced with AOM and treated with aqueous extract of the latex of E. tirucalli L., group 02 with rats induced with AOM without treatment with aqueous extract of the latex of E. tirucalli L., group 03 with rats without AOM induction and treated with aqueous extract of the latex of E. tirucalli L. and group 04 with rats without induction with AOM and without treatment with aqueous extract of the latex of E. tirucalli L.. The animals of the groups 01 and 02 were injected intraperitoneallly (IP), with AOM once a weekly for 02 weeks at a dose level of 12 mg/kg body weight. One week before the beginning of the administration of the carcinogen, it was administered solution daily by intragastric gavage, respectively, aqueous extract of latex of E. tirucalli L. 400mg/Kg and physiologic solution to 0,9%. The groups 03 and 04 were not induced with AOM and they received daily solution by gavage, respectively, aqueous extract of the latex of E. tirucalli L. 400mg/Kg and physiologic solution to 0,9%. All groups continued receiving daily solutions by intragastric gavage until the day established for euthanasia. All animals were sacrificed by diethyl ether inhalation 15th week, after AOM initiation or sterile solution injected IP. The animals were evaluated concerning to the weight, clinical alteration, presence of adenomas or colic tumors, and the presence of ACF and the number of crypts for each focus (multiplicity) in agreement with colic location, defined area distal, medial and proximal. We verified in the present study that the aqueous extract of E. tirucalli L. (400mg/kg) presented a significant decrease of the global number of ACF group 01 compared to the group 02 (p<0,001). Additionally, the multiplicity in these groups showed significant decrease (p<0,0001) in the number of ACF with 5 crypts/focus in the whole large intestine. This study suggests the aqueous extract of E. tirucalli L. has potential chemopreventive against colon carcinogenesis with inhibition of the formation of ACF in rats induced with AOM .

Skin cancer is a major health problem worldwide. It is the most common cancer in the United States and poses a significant healthcare burden. Excessive UVR exposure is the most common cause of skin cancer. Despite various precautionary measures to avoid direct UVR exposure, the incidence of skin cancer and mortality related to it remains high. Furthermore, the current treatment options are expensive and have side effects including toxicity to normal cells. Thus, a safe and effective approach is needed to prevent and treat skin cancer. Chemopreventive strategy using naturally occurring compounds, such as resveratrol, is a promising approach to reduce the incidence of UVR-induced skin cancer and delay its progression. This review highlights the current body of evidence related to chemopreventive role of resveratrol and its molecular mechanisms in UVR-induced skin carcinogenesis.

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