Gastric cancer is associated with NOS2-954G/C polymorphism and environmental factors in a Brazilian population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

ROBO1 deletion as a novel germline alteration in breast and colorectal cancer patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Screening da atividade antiproliferativa e investigação toxicológica in vitro e in vivo de compostos de coordenação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Low expression of human histocompatibility soluble leukocyte antigen-G (HLA-G5) in invasive cervical cancer with and without metastasis, associated with Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class Ib molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICCW)] and absence [ICC without metastasis (ICCWT)] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICCWT (n=52 patients) and ICCW (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies.

Evaluation of quality of life related to i-131 therapy in patients with well-differentiated thyroid cancer and emphasis in salivary morbidity: a follow up study after treatment

To evaluate the impact of iodine-131 therapy received during childhood and adolescence and correlate it with the quality of life in these patients. Methods: We studied 19 patients diagnosed with cancer in childhood or adolescence who underwent thyroidectomy and supplemental therapy with I-131. We also recruited a control group of healthy subjects with the same demographic parameters. All patients were subjected to a scintigraphy examination of the salivary glands, and were also asked to complete a questionnaire in order to assess their overall quality of life. In addition, a more specific questionnaire for patients with head and neck cancer was also given to all study participants. Results: The quantitative and qualitative analyses of the salivary glands showed functional deficits with greater involvement of the parotid gland for volume, concentration and excretion. The right submandibular gland showed significant changes for volume in the patient group. The questionnaires made it possible to observe significant differences between the patient and control groups for symptoms such as thick saliva, dry mouth and speech problems. Conclusion: In spite of being very effective and widely used, iodine radionuclide therapy is correlated with a lower quality of life in young people.

Modeling spatial distribution of cancer incidence in Cordoba, Argentina

Background: Cancer is the second leading cause of death in Argentina, and there is little knowledge about its incidence. The first study based on population-based cancer registry described spatial incidence and indicated that there existed at least county-level aggregation. The aim of the present work is to model the incidence patterns for the most incidence cancer in Córdoba Province, Argentina, using information from the Córdoba Cancer Registry by performing multilevel mixed model approach to deal with dependence and unobserved heterogeneity coming from the geo-reference cancer occurrence. Methods: Standardized incidence rates (world standard population) (SIR) by sex based on 5-year age groups were calculated for 109 districts nested on 26 counties for the most incidence cancers in Cordoba using 2004 database. A Poisson twolevel random effect model representing unobserved heterogeneity between first level-districts and second level-counties was fitted to assess the spatial distribution of the overall and site specific cancer incidence rates. Results: SIR cancer at Córdoba province shown an average of 263.53±138.34 and 200.45±98.30 for men and women, respectively. Considering the ratio site specific mean SIR to the total mean, breast cancer ratio was 0.25±0.19, prostate cancer ratio was 0.12±0.10 and lower values for lung and colon cancer for both sexes. The Poisson two-level random intercepts model fitted for SIR data distributed with overdispersion shown significant hierarchical structure for the cancer incidence distribution. Conclusions: a strong spatial-nested effect for the cancer incidence in Córdoba was observed and will help to begin the study of the factors associated with it.

Using Prior Information from the Medical Literature in GWAS of Oral Cancer Identifies Novel Susceptibility Variant on Chromosome 4-the AdAPT Method

Background: Genome-wide association studies (GWAS) require large sample sizes to obtain adequate statistical power, but it may be possible to increase the power by incorporating complementary data. In this study we investigated the feasibility of automatically retrieving information from the medical literature and leveraging this information in GWAS. Methods: We developed a method that searches through PubMed abstracts for pre-assigned keywords and key concepts, and uses this information to assign prior probabilities of association for each single nucleotide polymorphism (SNP) with the phenotype of interest - the Adjusting Association Priors with Text (AdAPT) method. Association results from a GWAS can subsequently be ranked in the context of these priors using the Bayes False Discovery Probability (BFDP) framework. We initially tested AdAPT by comparing rankings of known susceptibility alleles in a previous lung cancer GWAS, and subsequently applied it in a two-phase GWAS of oral cancer. Results: Known lung cancer susceptibility SNPs were consistently ranked higher by AdAPT BFDPs than by p-values. In the oral cancer GWAS, we sought to replicate the top five SNPs as ranked by AdAPT BFDPs, of which rs991316, located in the ADH gene region of 4q23, displayed a statistically significant association with oral cancer risk in the replication phase (per-rare-allele log additive p-value [p(trend)] = 2.5 x 10(-3)). The combined OR for having one additional rare allele was 0.83 (95% CI: 0.76-0.90), and this association was independent of previously identified susceptibility SNPs that are associated with overall UADT cancer in this gene region. We also investigated if rs991316 was associated with other cancers of the upper aerodigestive tract (UADT), but no additional association signal was found. Conclusion: This study highlights the potential utility of systematically incorporating prior knowledge from the medical literature in genome-wide analyses using the AdAPT methodology. AdAPT is available online (url: http://services.gate.ac.uk/lld/gwas/service/config).

Strategies and results of the oral cancer prevention campaign among the elderly in Sao Paulo, Brazil, 2001 to 2009

Objective. To describe the strategies and results obtained by the early diagnosis and prevention of an oral cancer campaign targeting the population aged 60 years or older developed since 2001 in the state of Sao Paulo. Methods. The main strategies used to develop the campaign were described based on the review of documents issued by the Health Ministry, National Cancer Institute, Sao Paulo State Health Department, Oncocentro Foundation of Sao Paulo, Sao Paulo City Health Department, School of Public Health at the University of Sao Paulo (USP), and Santa Marcelina Health Care Center. The impact of the campaign on the incidence of new cases of oral cancer in the target population was evaluated. Results. In 2001, 90 886 elderly were examined vs. 629 613 in 2009. The following strategies were identified: training of professionals, development of printed materials to guide municipal governments in developing the campaign and using standardized codes and criteria, guidelines for data consolidation, establishment of patient referral flows, practical training with a specialist at the basic health care unit after the follow-up examination of individuals presenting changes in soft tissues, and increase in the number of oral diagnosis services. Between 2005 and 2009, there was a significant reduction in the rate of confirmed cases of oral cancer per 100 000 individuals examined, from 20.89 to 11.12 (P = 0.00003). Conclusions. The campaign was beneficial to the oral health of the elderly and could be extended to include other age groups and regions of the country. It may also provide a basis for the development of oral cancer prevention actions in other countries, as long as local characteristics are taken into account.

Oral exposure to methylmercury modifies the prostatic microenvironment in adult rats

Methylmercury (MeHg) is an environmental pollutant that is highly toxic to the central nervous system. As its effects on male reproductive system are poorly understood, this study was carried out to analyse the effects of MeHg on the rat prostate. To evaluate the MeHg toxicity on ventral prostate, three groups of adult male Wistar rats received oral doses of 0.5, 1.0 and 3.0mg/kg MeHg, respectively, on a daily basis for 14days. A fourth group was used as a control. The prostate weight was decreased in rats treated orally with 0.5mg/kg MeHg compared to controls. Also, Hg concentration increased significantly in the prostate after treatments. There were reductions in serum testosterone levels and androgen receptor immunoreactivity in animals receiving 3.0mgMeHg/kg. The stereological data showed changes in the prostatic epithelial, stromal and luminal compartments which varied according to the different doses. Histopathological alterations, such as chronic inflammation, stratified epithelial hyperplasia and epithelial inflammatory reactive atypia, were observed in the 0.5mg/kg MeHg-treated group. Epithelial atrophy was observed in the 3.0mg/kg MeHg-treated group. In conclusion, the MeHg affects prostatic homoeostasis resulting in histopathological changes that may be relevant in the pathogenesis of prostatic disease.

Lung cancer symptoms and pulse oximetry in the prognostic assessment of patients with lung cancer

Abstract Background Medical oncologists continue to use performance status as a proxy for quality of life (QOL) measures, as completion of QOL instruments is perceived as time consuming, may measure aspects of QOL not affected by cancer therapy, and interpretation may be unclear. The pulse oximeter is widely used in clinical practice to predict cardiopulmonary morbidity after lung resection in cancer patients, but little is known on its role outside the surgical setting. We evaluated whether the Lung Cancer Symptom Scale and pulse oximetry may contribute to the evaluation of lung cancer patients who received standard anticancer therapy. Methods We enrolled forty-one consecutive, newly diagnosed, patients with locally advanced or metastatic lung cancer in this study. We developed a survival model with the variables gender, age, histology, clinical stage, Karnofsky performance status, wasting, LCSS symptom scores, average symptom burden index, and pulse oximetry (SpO2). Results Patient and observer-rated scores were correlated, except for the fatigue subscale. The median SpO2 was 95% (range: 86 to 98), was unrelated to symptom scores, and was weakly correlated with observer cough scores. In a multivariate survival model, SpO2 > 90% and patient scores on the LCSS appetite and fatigue subscales were independent predictors of survival. Conclusion LCSS fatigue and appetite rating, and pulse oximetry should be studied further as prognostic factors in lung cancer patients.

Trends in socioeconomic inequalities in cancer mortality in Barcelona: 1992–2003

Abstract Background The objective of this study was to assess trends in cancer mortality by educational level in Barcelona from 1992 to 2003. Methods The study population comprised Barcelona inhabitants aged 20 years or older. Data on cancer deaths were supplied by the system of information on mortality. Educational level was obtained from the municipal census. Age-standardized rates by educational level were calculated. We also fitted Poisson regression models to estimate the relative index of inequality (RII) and the Slope Index of Inequalities (SII). All were calculated for each sex and period (1992–1994, 1995–1997, 1998–2000, and 2001–2003). Results Cancer mortality was higher in men and women with lower educational level throughout the study period. Less-schooled men had higher mortality by stomach, mouth and pharynx, oesophagus, larynx and lung cancer. In women, there were educational inequalities for cervix uteri, liver and colon cancer. Inequalities of overall and specific types of cancer mortality remained stable in Barcelona; although a slight reduction was observed for some cancers. Conclusion This study has identified those cancer types presenting the greatest inequalities between men and women in recent years and shown that in Barcelona there is a stable trend in inequalities in the burden of cancer.

Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

Reorganization of secondary and tertiary health care levels: impact on the outcomes of oral cancer screening in the São Paulo State, Brazil

This study presents the strategies for prevention and early detection of oral cancer by means of screening in the elderly population of São Paulo, the richest and the most populous state of Brazil. This research was a retrospective longitudinal study based on the analysis of secondary data. The variables - number of participating cities, coverage of screening, and number of suspicious and confirmed cases of oral cancer - were divided into two periods: 2001-2004 and 2005-2008. Data were analyzed statistically by the chi-square test at 5% significance level. The implementation of a nationwide public oral health policy in 2004 and the reorganization of the secondary and tertiary health care were evaluated as mediator factors able to interfere in the achieved outcomes. From 2001 to 2008, 2,229,273 oral examinations were performed. There was an addition of 205 participating cities by the end of the studied period (p<0.0001). The coverage of oral cancer screening increased from 4.1% to 16% (p<0.0001). There was a decrease in the number of suspicious lesions (from 9% in 2005 to 5% in 2008) (p<0.0001) and in the rate of confirmed oral cancer cases per 100,000 examinations (from 20.89 in 2001 to 10.40 in 2008) (p<0.0001). After 8 years of screening, there was a decrease in the number of suspicious lesions and confirmed cases of oral cancer in the population. The reorganization of secondary and tertiary health care levels of oral care seems to have contributed to modify these numbers, having a positive impact on the outcomes of oral cancer screening in the São Paulo State.

RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.