Anàlisi de proteïnes portadores de l'antigen Sialil Lewis x (sLex) en cèl·lules tumorals del càncer de pàncrees

Abstract

Cancer is one of the most frequent diseases and one of the leading causes of death worldwide. Specifically, pancreatic cancer (PaC) has a very low survival rate due to the difficulty of diagnosing it in the early stages, among other factors. The PaC cells present alterations in their glycosylation pattern of their cell surface, such as an increase of Sialyl Lewis x (sLex) carbohydrate antigen which is related to their invasion capacity and metastasis. In the last stages of the sLex biosynthesis are requiered the enzymes α-2,3-sialyltransferases ST3Gal III or ST3Gal IV. In this project we have worked with two tumour cell lines; Capan-1 and BxPC3 and their respected silenced cell lines with shRNAs against the genes ST3GAL3 (sh7 and sh9) or ST3GAL4 (sh1 and sh4). The objective of this project was to analyze the differences of expression of sLex between the silenced lines respect to the control cell lines, and to determine the carriers of sLex that have decreased their content in sLex in the silenced lines. For these analyses, all the silenced (sh1, sh4, sh7 and sh9 of Capan-1 and sh1, sh7 and sh9 of BxPC3) and control cell lines have been cultivated. Two methodologies have been evaluated to obtain their protein cell lysate, and based on their quantification, we have compared the differences in sLex expression between silenced lines and control cell lines using anti-sLex in Western Blot (WB). The silenced cell lines show a decrease in sLex signal respect to the control lines, especially in high molecular weight proteins above 180kDa. In order to identify some of these proteins, we have analyzed the protein cell lysate using twodimensional electrophoresis. Based on the optimization of the two-dimensional electrophoresis, we have compared the blue Coomassie stain and the WB with sLex, and we have detected a group of protein that present a sLex signal in the control and in the silenced cell lines. Using their molecular mass and their isoelectric point we detected eight protein with sLex signal in all Capan-1 cell lines and eight proteins in all BxPC3 cells lines. In the case of Capan-1, we have identified three proteins with sLex signal in control cell lines but not in their silenced cell lines, and in the case of BxPC3 one protein with sLex signal which is not detected in their respected silenced cell lines