Orientadores: João Jose Fagundes, Wu Feng Chung / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T12:33:48Z (GMT). No. of bitstreams: 1 Rocha_AntonioAngelo_D.pdf: 7742775 bytes, checksum: 2cabe2d42821253fa63080904d8d166b (MD5) Previous issue date: 2010 / Resumo: A cicatrização das anastomoses intestinais depende de fatores locais e sistêmicos. A peritonite é fator importante que pode alterar a cicatrização das anastomoses no intestino. A hiperóxia hiperbárica (HBO) consiste na inalação de oxigênio a 100% em pressão superior à atmosférica. OBJETIVO: Analisar o efeito da oxigenoterapia hiperbárica sobre a resistência mecânica de anastomoses realizadas em cólon distal de ratos na presença de peritonite induzida por ligadura e punção cecal utilizando o teste biomecânico de Energia Total de Ruptura (ETR). MATERIAL e MÉTODO: Foram utilizados 45 ratos distribuídos em três grupos de 15 animais. No Grupo Controle (GC), realizou-se anastomose no cólon distal sem peritonite. No Grupo Peritonite (GP), realizou-se anastomose seis horas após a indução da peritonite por ligadura e punção cecal. No Grupo Câmara Hiperbárica (GCH), realizou-se anastomose seis horas após a indução da peritonite por ligadura e punção cecal. Os animais dos GC e GP foram mantidos em ar ambiente. Os animais do GCH foram colocados em uma câmara hiperbárica experimental para inalarem oxigênio a 100%, a duas atmosferas absolutas, durante 120 minutos, por quatro dias consecutivos. A eutanásia ocorreu no quinto dia do experimento. Todos os animais foram submetidos ao Teste de Resistência Biomecânico Energia Total de Ruptura (ETR). A Energia Total de Ruptura foi definida como a energia interna acumulada necessária para promover o rompimento do cólon após a imposição de uma força externa de tração. RESULTADOS: O Grupo Peritonite apresentou menor média de ETR que o Grupo Controle. Não houve diferença estatística entre o Grupo Peritonite e o Grupo Câmara Hiperbárica. CONCLUSÃO: A oxigenoterapia hiperbárica não alterou a resistência mecânica de anastomoses realizadas no cólon distal de ratos na presença de peritonite induzida por ligadura e punção cecal / Abstract: The cicatrization of bowel anastomosis depends on local and systemic factors. Peritonitis is an important factor that can alter the cicatrization of anastomosis in bowels. The HBO consists of inhaling oxygen at 100% under a pressure greater than the surrounding atmosphere. PURPOSE: To analyze the effect of hyperbaric oxygen therapy on the mechanical resistance of the anastomosis in the distal colon of rats in the presence of peritonitis induced by cecal ligation and puncture using the Total Energy Rupture biomechanical test (ETR). MATERIAL and METHOD: We used 45 rats divided into three groups of 15 animals. In the Control Group (CG) distal colon anastomosis was carried out without peritonitis. In the Peritonitis Group (GP), anastomosis was carried out six hours after the induction of peritonitis by cecal ligation and puncture. In the Hyperbaric Chamber Group (GCH), anastomosis was carried out six hours after induction of peritonitis by cecal ligation and puncture. The animals in the study and control groups were kept in environmental air. The GCH animals were placed in an experimental hyperbaric chamber to inhale 100% oxygen, at two absolute atmospheres for 120 minutes, for four consecutive days. Euthanasia occurred on the fifth day of the experiment. All animals were submitted to the Total Energy Rupture biomechanical test (ETR). The Total Energy Rupture was defined as the accumulated internal energy necessary to promote the rupture of the colon after the imposition of an external force of traction / Doutorado / Pesquisa Experimental / Mestre em Cirurgia

Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.

Colonic epithelial glycoproteins were histochemically studied in rats during the induction of colorectal cancer with 1,2,-dimethylhydrazine-diHCl (DMH). 310 male Wistar rats were separated into 3 groups: 10 control rats were sacrificed without treatment, 150 rats were given weekly subcutaneous injections of the carcinogen and 150 rats were given sham Injections on the same schedule. Groups of rats (10 control and 10 treated) were sacrificed at various time intervals in an effort to obtain a large number of pre-neoplastic rat colons to be used to determine whether histochemical changes could be detected prior to morphologic change. Three histochemically distinct regions in the Wistar rat distal colon have been described using recently developed techniques. Two major histochemical changes have been shown to occur prior to malignancy. Change 'A' represented a decrease or loss of sulphate from the glycoproteins; this was the earliest and predominant change and has been shown to occur prior to any identifiable morphologic change. Change 'B' represented a decrease or loss of sulphate occurring in the same cells as a decrease or loss of side chain 0-acetylation of sialic acid residues; this change occured later and to a lesser extent than change 'A'. The data suggests that histochemical change may be a premalignant marker, however, further investigations are necessary to firmly establish this conclusion. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

No description available.

Ample studies have shown that perturbation of metabolic phenotype is correlated with gene mutation and pathogenesis of colorectal cancer (CRC) and rheumatoid arthritis (RA). Mass spectrometry (MS)-based metabolomics as a powerful and stable approach is widely applied to bridge the gap from genotype/metabolites to phenotype. In CRC suffers, KRAS mutation accounts for 35%-45%. In previous study, SLC25A22 that encodes the mitochondrial glutamate transporter was found to be overexpressed in CRC tumor and thus to be essential for the proliferation of CRC cells harboring KRAS mutations. However, the role of SLC25A22 on metabolic regulation in KRAS-mutant CRC cells has not been comprehensively characterized. We performed non-targeted metabolomics, targeted metabolomics and isotope kinetic analysis of KRAS-mutant DLD1 cells with or without SLC25A22 knockdown using ultra-high performance liquid chromatography (UHPLC) coupled to Orbitrap MS and tandem MS (MS/MS). In global metabolomics analysis, 35 differentially regulated metabolites were identified, which were primarily involved in alanine, aspartate and glutamate metabolism, urea cycle and polyamine metabolism. Then targeted metabolomics analysis on intracellular metabolites, including tricarboxylic acid (TCA) cycle intermediates, amino acids and polyamines, was established by using LC-MS/MS coupled with an Amide BEH column. Targeted metabolomics analysis revealed that most TCA cycle intermediates, aspartate (Asp)-derived asparagine, alanine and ornithine (Orn)-derived polyamines were strongly down-regulated in SLC25A22 knockdown cells. Moreover, the targeted kinetic isotope analysis using [U-13C5]-glutamine as isotope tracer showed that most of the 13C-labeled TCA cycle intermediates were down-regulated in SLC25A22-silencing cells. Orn-derived polyamines were significantly decreased in SLC25A22 knockdown cells and culture medium. Meanwhile, accumulation of Asp in knockdown of GOT1 cells indicated that oxaloacetate (OAA) was majorly converted from Asp through GOT1. Exogenous addition of polyamines could significantly promote cell proliferation in DLD1 cells, highlighting their potential role as oncogenic metabolites that function downstream of SLC25A22-mediated glutamine metabolism. SLC25A22 acts as an essential metabolic regulator during CRC progression as promotes the synthesis of TCA cycle intermediates, Asp-derived amino acids and polyamines in KRAS-mutant CRC cells. Moreover, OAA and polyamine could promote KRAS-mutant CRC cell growth and survival. Rheumatoid arthritis (RA) is a chronic, inflammatory and symmetric autoimmune disease and a major cause of disability. However, there is insufficient pathological evidence in term of metabolic signatures of rheumatoid arthritis, especially the metabolic perturbation associated with gut microbiota (GM). Based on consistent criteria without special diet and therapeutic intervention to GM, we enrolled 50 RA patients and 50 healthy controls. On basis of the platform of UHPLC-MS and GC-MS, were performed for the non-targeted metabolomics to investigate alterations of endogenous metabolites in response to RA inflammation and interaction with GM. 32 and 34 significantly changed metabolites were identified in urine and serum of patients with RA, respectively. The altered metabolites were identified by HMDB, METLIN database or authentic standards, and mostly metabolites were attributed into tryptophan and phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, aminoacyl-tRNA biosynthesis and citrate cycle. To obtain alterations of more components in tryptophan and phenylalanine metabolism, we developed and validated a targeted metabolomics method of 19 metabolites by using LC-QqQ MS. Combining the results of targeted metabolomics with global metabolomics, significantly up-regulated kynurenine (KYN), anthranilic acid (AA) and 5-hydroxylindoleacetic acid (HIAA) simultaneously in urine and serum was found to implicate the activation of tryptophan metabolism under the condition of RA, which acted pro-inflammatory roles in inflammation and was closely correlated with GM. IDO/TDO functioned as a pro-inflammation mediator was overexpressed in RA patients. Urinary kynurenic acid and serum serotonin that have impacts on anti-inflammation in immune system were down-regulated in RA patients. The levels of phenylacetic acid and phenyllactic acid serving as a pro-inflammatory and an anti-inflammatory agent, respectively, increased in serum of patients with RA. Moreover, certain essential amino acids (EAAs), and mostly conditional EAAs were decreased in RA patients, which have been reported to inhibit cell proliferation of immune cells. In particular, deficiency of branched chain amino acids (BCAAs, valine and isoleucine) was observed in serum of patients with RA, which may lead to muscle loss and cartilage damage. The specificity of all altered metabolites resulted from RA was considerably contributed through the GM-derived metabolites. The findings revealed that GM-modulated RA inflammation was mainly resulted from tryptophan and phenylalanine metabolism, and amino acid biosynthesis, which may provide more information for better understanding the RA mechanism.

Colon cancer is one of the most common cancers in both the United States (US) and throughout the world. Over the last 30 years, despite the development of multiple classes of effective anti-tumor agents, colon cancer has consistently remained the second leading cause of mortality amongst all cancers and is today responsible for over 50,000 deaths a year in the US alone. Among the greatest challenges to the successful treatment of colon cancer is its heterogeneity in terms of drug-sensitivity, whereby it is often difficult to identify which patients will benefit from a specific class of anti-tumor agents before treatment has begun. It is therefore imperative to identify predictive biomarkers that can be leveraged to distinguish which colon tumors are most likely to respond to individual anti-cancer drugs. This will help develop new therapeutic algorithms that can maximize patient survival by rapidly matching individual patients with the specific treatment combinations that are most likely to benefit them as well as sparing them the toxicities from drugs that would be ineffective. Previous studies have reported that human colon carcinomas lacking expression of the caudal-type homeobox 2 (CDX2) transcription factor can be leveraged as a predictor of benefit from adjuvant chemotherapy containing 5-fluorouracil (5-FU). Lack of CDX2 expression associates with microsatellite instability (MSI), as well as several histopathological and molecular features that associate with exceptionally poor prognosis such as poor differentiation, lympho-vascular invasion, and BRAF mutation. However, the molecular mechanisms linking lack of CDX2 expression with increased drug sensitivity are currently unknown. In the first section of this study, we conducted a high throughput screen (HTS) aimed at identifying clinically approved anti-tumor drugs that display selective activity against colon carcinomas lacking CDX2 expression (CDX2-negative). The results of our screening, which compared an isogenic pair of CDX2+/+ and CDX2-/- cell lines generated by genetic inactivation of CDX2 using CRISPR/Cas9 constructs, revealed that CDX2-negative colon cancer cells display increased sensitivity to anti-tumor drugs that are substrates of the ATP binding cassette sub-family B member 1 (ABCB1) transporter. ABCB1 is a drug-efflux protein known for its capacity to extrude multiple classes of anti-tumor agents from the cytoplasm, therefore contributing to drug-resistance in cancer cells. Importantly, analysis of CDX2 and ABCB1 expression in two independent gene-expression databases (NCBI-GEO: n=2115; TCGA: n=478) revealed that a lack of CDX2 expression is invariably associated with lack of ABCB1 expression in human primary colon carcinomas. Furthermore, our molecular studies revealed that forced expression of CDX2 in human CDX2-negative colon cancer cells was capable of inducing expression of ABCB1, while genetic inactivation of CDX2 in human CDX2-positive cancer cells using CRISPR/Cas9 constructs resulted in loss of ABCB1 expression, thus establishing CDX2 as a direct mechanistic regulator of ABCB1 expression. Amongst all of the anti-tumor drugs identified as being ABCB1 substrates with preferential activity against CDX2-negative colon cancer cells, we observed that paclitaxel was the FDA-approved drug with the greatest degree of selectivity with a 10-fold difference in IC50. When tested in vivo against a collection of human patient derived xenograft (PDX) lines representative of both CDX2-negative and CDX2-positive colon carcinomas, paclitaxel displayed selective activity against CDX2-negative models, often inducing volumetric regression of established lesions. Our study, therefore, identified paclitaxel as a clinically approved anti-tumor agent that should be investigated for use in the treatment of CDX2-negative colon carcinomas. In the second portion of our study, we sought to conduct a preliminary evaluation of the possibility of using immune checkpoint inhibitors (ICIs) for the treatment of CDX2-negative colon carcinomas. ICIs have been shown to display substantial anti-tumor activity against colon carcinomas with microsatellite instability (MSI) and against epithelial malignancies over-expressing the immune-suppressive molecule PD-L1/CD274. Because CDX2-negative tumors are enriched for MSI and high levels of PD-L1/CD274, they are predicted to include a subgroup that is responsive to ICIs. However, not all MSI tumors respond to ICIs and, contrary to the majority of MSI tumors, the subgroup of MSI tumors characterized by a CDX2-negative phenotype is often associated with poor prognosis. Because the clinical activity of ICIs is dependent upon expression of class-I HLA molecules by tumor cells, we decided to evaluate whether CDX2-negative tumors were associated with inactivating mutations in class-I HLA genes. Our attention focused on a highly conserved poly-cytosine repeat region in the coding sequence of HLA-A (c.621_627) and HLA-B (c.621_626) genes. Because this sequence fulfilled the molecular definition of microsatellite, we predicted it to be highly susceptible to frameshift mutations (insertions or deletions) in MSI colon tumors. Indeed, a search across three independent genetic databases (TCGA, COSMIC, EBI) confirmed that this highly conserved poly-cytosine repeat region was targeted by recurrent and deleterious mutations in at least one HLA-A or HLA-B allele of at least 13% (n=21/156) of human MSI colon tumors, as compared to 0.3% (n=2/770) of human colon tumors with a microsatellite stable (MSS) phenotype (p<0.0001). Among tumors assessable for CDX2 expression, this specific type of class-I HLA mutations was more frequent among CDX2-negative (12%; n=6/49) as compared to CDX2-positive (1.5%; n=5/340) colon tumors (p<0.001), but was similar within MSI CDX2-negative (21%; n=6/28) and MSI CDX2-positive (17%; n=5/30) subgroups. In summary, this work achieved two main results: 1) it identified paclitaxel, a clinically approved anti-tumor drug, as a new treatment option for patients with CDX2-negative colon cancers, which represents an extremely aggressive subgroup of colorectal malignancies; 2) it revealed that, in human MSI colon tumors, class-I HLA genes are prone to recurrent frameshift mutations in a genomic hotspot, mutations that are likely to associate with tumor resistance to ICIs and that they are therefore likely to represent a new class of actionable predictive biomarkers for both MSI and CDX2-negative colon carcinomas. These findings will help advance our understanding of colon cancer biology, and hopefully improve treatment algorithms for the clinical management of colon cancer patients.

Colorectal cancer (CRC) is the third most prevalent and second leading causes of cancer-associated deaths globally. Over the last few decades, ent-kaurane diterpenes have been widely investigated for their anticancer potentials. Flexicaulin A (9) is a naturally occurring ent-kaurane. Previously, our lab modified the structure of 9 by replacing the C-11 hydroxyl group with carbonyl group and obtained a novel compound oxoflexicaulin A (11). However, anticancer activities and mechanistic pathway of these two compounds are yet to be explored. In the current thesis, we evaluated the cytotoxicity of compounds 9 and 11 in A549 lung, A375 melanoma, PANC1 pancreatic, HCT-116 and HT-29 colon cancer and 293T human embryonic kidney cells as well as compared the activity with a number of known natural ent-kauranes to describe their structure-activity relationship. Our study found that the presence of α, β-unsaturated ketone groups in ent-kaurane structure acted as the pharmacophore. The replacement of C-11 hydroxyl group by carbonyl in 9 (IC50: 3.68 µM) gives a novel potent anticancer compound 11 (IC50: 0.77 µM). Considering the novelty and superior activity of 11, its mechanistic pathway was studied in HCT-116 cells and compared with the natural scaffold 9. Flow cytometry analysis by Annexin V/PI staining along with fluorescent staining by DAPI showed that both compounds induced apoptosis in HCT-116 cells. Induction of apoptosis is mediated through up-regulation of tumor suppressor protein p53 and pro-apoptotic protein Bax, Bak and puma as well as promoting the cleavage of PARP while down-regulation of anti-apoptotic protein Bcl-2 and PARP. Apart from their effect on apoptosis, compounds 9 and 11 stimulated the event of senescence, a process of cellular aging, as confirmed by β-galactosidase assay. Induction of senescence is related with up-modulation of p21 and p27 while down-modulation of p16, Rb and its transcription factor E2F1. Moreover, immunofluorescence staining showed translocation of p21 and p27 from the cytoplasm to nucleus after treatment with 9 and 11. Further study found that the two ent-kauranes inhibited the protein level of two NF-κB sub-units p65 and p50 in the nucleus as well suppressed the cytoplasmic level of NF-κB inhibitor IkB-α. Both compounds also inhibited the expression and phosphorylation of STAT3 in the cytoplasm and nucleus, so as for the expression and phosphorylation of Src, an up-stream kinase of STAT3. In xenograft nude mice model, compound 11 remarkably inhibited tumor growths (volume and size) but the body weights of the mice were also reduced (p < 0. 05). Therefore, we designed to synthesis a series of prodrug analogs from 11 by adding acetal protecting group to reduce the toxicity. One of the prodrug (37) significantly attenuated the tumor volume and size (p < 0.05) at 50 mg/kg without any toxicity (p > 0.05). The prodrug 37 is actually released as compound 11 in the mice due to its cleavage in the acidic microenvironment of tumor. Taken together, antitumor effect of compound 11 in CRC model is supposed to be mediated through induction of apoptosis and senescence via modulation of NF-κB and STAT3 signaling pathway. Keywords: Colorectal cancer, ent-kaurane, flexicaulin A, oxoflexicaulin A, cytotoxicity, anticancer, apoptosis, pathway, NF-κB, STAT3.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Colorectal cancer (CRC), the third leading cause of cancer-related death in the U.S., could largely be prevented if more people had polyps removed via colonoscopies. Embarrassment has been identified as one important barrier to colonoscopy, but little is known about embarrassment in this context. Further, there is no instrument available to measure this construct. Therefore, the purpose of this study was to develop a reliable and valid instrument to measure colonoscopy-related embarrassment. The study aims were to: 1) estimate reliability and validity of a new instrument, the Colonoscopy Embarrassment Scale (CES); 2) examine relationships among demographic/personal characteristics, health beliefs, and CES scores; 3) examine relationships among demographic/personal characteristics, physician recommendation, health beliefs, and colonoscopy compliance; and 4) evaluate participants’ perceptions of aspects of having a colonoscopy that are most embarrassing and their suggestions for reducing embarrassment. The Health Belief Model and Transtheoretical Model of Change provided theoretical support for this study. Participants were HMO members aged 50-65 years (n=234). Using a cross-sectional, descriptive research design, data were collected using a mailed survey. The response rate was 56%. Data were analyzed using independent samples t-tests, correlations, Chi Square, and regression. Results showed that the six-item CES had internal consistency (Cronbach’s alpha of .89) and construct validity. Lower income, higher BMI, lower CRC knowledge, higher barriers, and lower self-efficacy were related to higher CES scores (or more embarrassment). Higher CRC knowledge, lower barriers, higher self-efficacy, and a physician recommendation for the test were related to higher compliance with colonoscopy. Lower barriers, higher self-efficacy, and a physician recommendation were predictive of compliance with colonoscopy. In conclusion, embarrassment is a significant barrier to colonoscopy, yet there are steps that can be taken to reduce embarrassment such as increasing privacy and limiting bodily exposure. The CES is a tool that can be used to measure colonoscopy-related embarrassment and the results could be used in developing further interventions to reduce embarrassment, leading to increased colonoscopies and lower mortality.

The hypothesis was evaluated that lower dietary consumption of carotene or retinol or lower serum levels of beta carotene or retinol are associated with development of colonic adenomas. To evaluate this hypothesis, selected patients who were to undergo colonoscopy to determine polyps status were asked to undergo a battery of tests to assess nutritional status. These tests included a dietary and demographic questionnaire, serologic assessment of beta carotene and retinol, and a dark adaptation test. One hundred male subjects were evaluated. Fifty-seven were found to have colonic adenomatous polyps. Cases and controls appeared to be well matched for demographic characteristics. There were no statistically significant differences for any nutritional parameter between cases and controls, but cigarette smoking was more prevalent among cases than controls (p<0.05). Because nonsignificant negative associations with colonic adenomas were observed for some of the nutritional parameters, it is concluded that additional subjects should be studied, as planned. / M.S.