Disseny experimental per a la caracterització de models cel·lulars amb els gens ST3GAL3 i ST3GAL4 silenciats

Abstract

Cancer is nowadays a global health problem constantly increasing, being the second cause of death in developed countries. Pancreatic adenocarcinoma, often referred as pancreatic ductal adenocarcinoma (PDAC), is the fourth leading cause of cancer death in Europe and EEUU with a mortality tax greater than 90%, even though it only represents a 3-6 % of cancer cases within population. The glucidic alteration associated to tumoral processes is a well described feature. In this context, the overexpression of the glucidic antigens Sialyl Lewis X (SLeX) and Sialyl Lewis A (SLeA) in PDAC, significally enhances the aggressiveness of the pathology (more migration, invasion and metastasis capability). The synthesis of these antigens is complex and involves the sialyltransferases (STs) and other enzymes. STs are enzymes that place sialic acids (Sias) in terminal position of glucidic chains. The sias are diverse in structure, but they all are negatively charged, promoting de-adhesion of tumour cells by charge repulsion (and thus, contributing to metastasis formation). In this work, the attention is centred on SLeX and SLeA antigens, and on ST implicated in their formation: ST3Gal III and ST3Gal IV. In this line, a lack of these enzymes should directly mean a reduction of the antigens and the metastatic behaviour of the cells. The starting point is having different cell lines of PDAC: parental (non-silenced), and silenced in ST3GAL3, ST3GAL4 or in both genes. Thus, the objective is to define an experimental design by searching bibliography, allowing to, firstly, characterise the silencing, and secondly, characterise the metastatic phenotype. Attempting to achieve this objective, different analyses have been proposed. The first part includes: quantification of protein expression levels (by western blot, in order to quantify the STs), quantification of the antigens (by flow cytometry against the SLeX and SLeA structures) and a viability assay (MTT), with the intention to check out if lack of STs or SLeX and/or SLeA antigens aren’t a significant compromise for cell viability. These analyses make up the silencing characterisation