Investigation of molecular and genetic mechanisms af anastasis in non-small cell lung cancer

Can a dying cell escape the brink of cell death after reaching the generally accepted 'points of no return'? This question constitutes the first step of the mechanism of anastasis, but also led to the emergence of this thesis. Anastasis, which has been described in the literature as a reversal of apoptosis, begins with the removal of the inducing agent from the environment. More work is needed on the purpose of anastasis and how it works, as it is a newly discovered process. This thesis study reveals that A549 cells can recover from Paclitaxel-induced apoptosis. In the first stage, in order to convert A549 cells into anastatic cells, the maximum toxic concentration of Paclitaxel was determined by creating growth curves after the application of Paclitaxel with SRB cell viability analysis. In addition, the results were confirmed by ATP cell viability analysis, which is a more sensitive method. In the second step, it was determined at what time interval anastatic cell transformation occurred by using the maximum toxic concentration of Paclitaxel. Early and late stages of apoptosis were determined by triple fluorescent staining (Hoechst 33342, Propidium iodide and Annexin-V) and Caspase 3/7 enzyme activity was determined with a luminometric caspase measurement kit. In addition, the presence of Caspase 3/7+ and Propidium iodide- and Caspase 3/7+, Propidium iodide+ cell populations at the specified times and concentrations was confirmed using flow cytometry. In this way, it has been established at what time and concentrations paclitaxel induces apoptotic cell death in the majority of the cell population (>80%). In the final stage, after applying the paclitaxel to A549 cells at the specified times and concentrations, it was removed from the environment and anastatic cell transformation was investigated by western blot at the protein level and RT-PCR method at gene level. As a result of this thesis study, it was shown that A549 cells reverse Paclitaxel-induced apoptotic cell death. It has been shown in vitro that Non-Small Cell Lung Cancer cells (A549) come back by reversing Paclitaxel-induced apoptotic cell death, and the presence of the mechanism of these returning cells (anastatic) at the protein and gene levels.