Lipid droplets (LDs) are dynamic organelles found in most eukaryotic cells. They play an important role in the regulation of lipid metabolism, uptake, distribution, storage, and utilization of fatty acids within cells. LDs help maintain redox homeostasis, membrane integrity, modulate cellular response to stress and protect cells against lipotoxicity. Their synthesis and breakdown is regulated according to the energy needs of the cell. Lipolysis and lipophagy are the main pathways for LD degradation. Adipose triglyceride lipase (ATGL) is the primary cytosolic hydrolase catalysing the first step of lipolysis, facilitating the release of fatty acids from LDs.
Ferroptosis is a form of cell death induced by lipid peroxidation. LDs influence the cellular distribution of polyunsaturated fatty acids, which are required for the process of ferroptosis. ATGL significantly contributes to intracellular lipid trafficking by releasing fatty acids from LDs. However, the connections between LDs and ferroptosis have not been fully elucidated. We investigated the role of LD lipolysis in modulating ferroptosis using a novel inhibitor of the human ATGL enzyme, NG-497. We hypothesized that the NG-497 inhibitor would have a protective role against ferroptosis by preventing the release of polyunsaturated fatty acids from LDs. The aim of this master’s thesis was to evaluate the toxicity of inhibitor NG-497, examine its impact on LD degradation under various conditions, and assess its effect on the sensitivity of cancer cells to ferroptosis.
First, we evaluated the ability of the NG-497 inhibitor to inhibit lipolysis using flow cytometry and confocal microscopy. Our finding demonstrated that the inhibitor increases the LD content in cancer cells. Colocalization between lysosomes and LDs increased with the inhibition of lysosomal acid lipase, while simultaneous inhibition of ATGL and lysosomal acid lipase did not alter the effect observed with inhibition of lysosomal acid lipase. We then assessed the impact of NG-497 on cancer cell sensitivity to RSL3-induced (RAS selective lethal 3) ferroptosis. Contrary to our hypothesis, our findings indicate that NG-497 does not alter ferroptosis sensitivity. Our results will contribute to the better understanding of the connections between LDs and ferroptosis.
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