DUGi

Optimització de metodologies per detectar la mort cel·lular per apoptosi

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Cancer is one of the deadliest diseases nowadays, so developing effective therapies against it has become an increasingly important need in our society. These treatments, aside from the fact that they must be directed exclusively against cancer cells, they also need to induce a type of controlled death to avoid their side effects as much as possible. Apoptosis, a type of programmed cell death, is characterized by being highly regulated, occurring individually and without affecting the surrounding healthy cells. Although this process can be identified with various techniques, no universal method is known to detect it. During this study, three apoptosis detecting methods have been compared and, in conclusion, it has not been possible to determine a single protocol that is completely effective for all cell lines. To determine the efficiency of these methods, apoptosis has been induced in the A549 and HeLa lines with doxorubicin and cisplatin, two drugs with known anti-tumour action. For the A549 line, successful results have been obtained by western blot when analyzing the activation of caspase 3, one of the main effector proteins of apoptosis and its biomarker. Additionally, when analyzing the same line by flow cytometry, positive results were also obtained regarding the activation of programmed cell death. Regarding the HeLa line, despite having observed isolated apoptotic populations with the last mentioned technique, it was not possible to correctly identify the activation of caspases by means of western blot. Based on the third methodology studied, an enzymatic assay for the quantification of caspase activity, no conclusion could be reached, as the results obtained were not considered valid. This project proposes new lines of research to find a single standardized protocol to detect programmed cell death in culture, which could be useful for evaluating the effectiveness of new anticancer drugs in development

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