As neoplasias malignas são responsáveis por milhões de mortes todos os anos, sendo que os problemas acarretados por tumores gástricos representam a terceira causa de morte por câncer a nível mundial. Um dado preocupante em relação ao câncer gástrico é em relação a sobrevida global de 5 anos que é de apenas 30%, aproximadamente. Um dos principais fatores que acarretam em taxas tão alarmantes é que em estágios iniciais o câncer gástrico é assintomático, fazendo com que o seu diagnóstico ocorra normalmente em estágios mais tardios. Além disso, prognósticos ruins mesmo em estágios iniciais da doença são comuns neste tipo de câncer, principalmente devido ao alto índice de resistência das células tumorais aos tratamentos convencionais. Com a necessidade de novos biomarcadores e novas modalidades de tratamentos, o estudo do secretoma de células tumorais vem sendo amplamente explorado. Este tipo de estudo é utilizado como meio de identificar novas moléculas que possam ser utilizadas em diagnósticos mais precoces assim como novos alvos terapêuticos. Por isso, o objetivo desse trabalho foi analisar o perfil do secretoma de células tumorais derivadas de linhagens de câncer gástrico (ACP02 e ACP03), em comparação com uma linhagem de mucosa gástrica normal (MN01), buscando novas moléculas que possam servir de biomarcadores, bem como fonte para novos alvos terapêuticos. Para o alcance desse objetivo o meio condicionado das diferentes linhagens celulares foi coletado, e as proteínas presentes foram concentradas através de ultrafiltração e identificadas através de análises por Q-Tof LC/MS/MS. A análise de espectrometria de massas identificou 333 proteínas secretadas, sendo 86 exclusivamente secretadas por ambas as linhagens de câncer gástrico. Após a identificação, as proteínas foram analisadas através de biologia de sistemas e, devido a sua importância, as proteínas PDIA6, GDF15 e HSP47 foram escolhidas para análises por Western blot e qPCR. A análise de Western blot confirmou que as proteínas GDF15 e HSP47 são secretadas apenas pelas linhagens de adenocarcinomas gástricos. Porém quando analisamos a proteína PDIA6 não conseguimos comprovar a sua secreção diferencial pelas linhagens de adenocarcinomas. Com estes resultados, resolvemos analisar a proteína HSP47 como possível alvo terapêutico, através do efeito do seu silenciamento nas linhagens de câncer gástrico pela técnica de CRISPR. Estas análises demonstraram que o silenciamento da proteína HSP47 reduziu a capacidade migratória, invasiva e de formação de esferas nas células tumorais. Em síntese, os resultados demonstraram que a HSP47 pode ser um possível alvo terapêutico no tratamento do câncer gástrico, e que HSP47 e GDF15 possuem potencial para serem utilizadas como biomarcadores devido a sua secreção diferencial pelas células tumorais. / The malignant neoplasms are responsible for millions of deaths every year, and the gastric cancer is the third leading cause of cancer deaths worldwide. An alarming finding regarding gastric cancer is the five-year survival rate of only 30%. One of the main problems related to gastric cancer is that it is asymptomatic in the initial phases of the disease, making their diagnosis usually occur in later stages. Also, poor prognoses even in the early stages of the disease are frequent in this type of cancer, mainly due to the high resistance of tumor cells to conventional treatments. The study of the secretome of tumor cell lines has been extensively explored to discover new biomarkers and new modalities of treatments, that can be used in earlier diagnoses as well as new therapeutic targets. Then, the present study aimed to characterize the secretome of two human gastric cancer cell lines (ACP02 and ACP03) in order to compare their differences concerning the non-neoplastic gastric cell line (MN01). For this purpose, we collected the conditioned medium of these cell lines and concentrated the secreted proteins by ultrafiltration. After that, the identification of the proteins was made by ultra-performance Q-Tof LC/MS/MS. Mass spectrometry-based proteomics identified 333 proteins in the three cell lines of which 86 proteins are identified exclusively from the two carcinoma cell lines. After the identification, the proteins were analyzed through system biology and, due to their importance, the proteins PDIA6, GDF15 e HSP47 were chosen for western blot and qPCR analyzes. The Western blot analysis showed that GDF15 and HSP47 are secreted only by the tumor cell lines. However, when we analyzed de PDIA6 protein, we are not able to confirm the differential secretion by the tumor cell lines. With these results, we decided to analyze the HSP47 protein as a possible therapeutic target, through the effect of its silencing on gastric cancer lines by the CRISPR technique. These analyses showed that HSP47 silencing decreases the migration, invasion, and sphere forming capacity in gastric tumor cells. In summary, the results demonstrated that the HSP47 protein may be a possible therapeutic target in the treatment of gastric cancer and that HSP47 and GDF15 proteins have potential to be used as biomarkers, due to their differential secretion by tumor cells.

Made available in DSpace on 2014-06-11T19:30:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2007Bitstream added on 2014-06-13T20:01:07Z : No. of bitstreams: 1 ferrasi_ac_dr_rcla.pdf: 1030993 bytes, checksum: 9a0fbcc4634e2697322fd343cddaa074 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Gastric cancer (GC) is one of the most common cancer types and it is associated with high mortality frequencies. Although a decrease in the worldwide incidence is observed, the prognosis of this disease still remains poor, mainly when the diagnosis is carried out at advanced stages. Recent evidences have identified DNA methylation as an important mechanism for tumor suppressor gene inactivation. Helicobacter pylori infection is considered one of the most important etiological factors and the CagA gene is associated with more severe pathologies including cancer. Likewise, EBV is another infectious agent that has been associated with gastric carcinoma in at least 10% of the cases. In this study, we determined the promoter methylation status of the CDH1, DAPK, COX2, hMLH1 and CDKN2A and MSI frequency in 89 primary gastric carcinomas and correlated the findings with the presence of H. pylori and EBV infections and also with clinicopathological features of gastric carcinomas. COX2 was the most frequently hypermethylated gene (63.5%) in these patients, followed by DAPK (55.7%), CDH1 (51%), CDKN2A (36%) and hMLH1 (30.3%). In this study, MSI was correlated with hMLH1 methylation, as shown before, and there was an inverse correlation between DAPK hypermethylation and MSI. Also, MSI was inversely correlated with H. pylori CagA+, providing new evidence for the association of MSI and better prognosis.