Cancer cells: a crosstalk between general metabolism and sensitivity to apoptotic signals

• Autor(a) principal: Fernandes, Andreia Silva
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10348/8800
• topic: Cancer; mitochondria; metabolism; apoptosis; ROS; Redox potential

The transformation of a normal cell into a cancer cell is a pathological process involving several stages, which modifies cellular metabolism and supports: rapid generation of ATP, high rate of macromolecules biosynthesis and maintenance of the appropriate cellular redox state. Mitochondria play a central role in the eukaryotic cell because of their relevance in biosynthetic and bioenergetic metabolisms. Additionally, mitochondria are also responsible for the production and/or control of various types of signals (including molecules), involved in cellular signalling processes that modulate various aspects of the functional organization of cells and their interaction with neighbouring cells. For example, this organelle is particularly associated with the processes of cell death by apoptosis. In this context, the development and progression of oncologic disease is inevitably associated with changes in metabolic pathways/signalling that involve mitochondria. Two models of human cancer cells, notably A549 cells (representative of non-small cell lung cancer) and AGS cells (representative of stomach cancer promoted by intestinal type cells), are used in the present work to investigate the relationship between metabolic parameters and sensitivity of cancer cells to apoptotic stimuli. This aspect is fundamental to understanding the mechanisms used by cancer cells to develop resistance to conventional chemotherapy and also to develop new and more effective therapeutic strategies. The results obtained show that these two cell lines show distinct profiles of growth curves. The cell proliferation rate (inversely proportional to the doubling time) of A549 cells is slightly higher than AGS cells. However, when compared to A549 cells, AGS cells exhibit higher intracellular glycogen content, higher MTT reduction ability and much higher mitochondrial functionality, as revealed by the parameters that characterize mitochondrial respiration (basal respiration, respiration associated with synthesis of ATP and maximum breathing capacity). Additionally, the lipid composition of the membranes of these cells also depends on the cell type. When normalized by the amount of protein, A549 cells exhibit higher lipid content (phospholipids + cholesterol) but lower glycolipid content than AGS cells. Analysis of the fatty acid profiles revealed that A549 cells exhibit lower relative abundance of saturated fatty acids, higher abundance of monounsaturated fatty acids and a higher ratio of Omega6/Omega-3 polyunsaturated fatty acids to those of AGS cells. The characterization of the oxidation-reduction cell state was evaluated by the GSH/GSSG ratio. The activity of the enzymatic antioxidant defences and the rate of production of reactive oxygen species follow a pattern consistent with the metabolic differences presented by the two cell lines. Thus, AGS cells exhibit Catalase and Superoxide dismutase (SOD) activities superior to A549 cells, despite their r viii intracellular compartments are, in average, a more oxidized state, as suggested by the lower ratio GSH/GSSG. Notwithstanding these differences, the ROS production rate is similar in both cell lines. Considering the effect of increasing concentrations of staurosporine on cell viability, AGS cells (IC50 = 20.5 nM) are shown to be significantly more sensitive than the A549 cells (IC50 = 108.0 nM) to this apoptotic agent. Staurosporine, at the concentration of IC50 for each cell line, promotes a significant decrease in the electrical potential across the mitochondrial inner membrane (ΔΨm) in both cell lines without affecting ROS generation. Additionally, the A549 cell line was shown to exhibit ΔΨm significantly higher than the AGS cell line, exhibiting more hyperpolarized mitochondria. The impact of the destruction of the extracellular matrix (through trypsin activity) in the oxidationreduction cell, ΔΨm and ROS production was also evaluated in both cell lines. The trypsinization process destroys the extracellular matrix and transforms adherent cells into mobile/floating cells, promoting in both cell lines a significant increase in ROS production. These effects are more evident in A549 cells. The increase in ROS production rate is accompanied by a decrease in the GSH/GSSG ratio and an increase of ΔΨm; and again, these effects are also more evident in A549 cells. Together, the results support the idea that the higher tumour aggressiveness of A549 cells, compared to AGS cells, detected by the higher rate of cell proliferation and the greater the ability to evade apoptosis (less sensitivity to staurosporine) are reflex of the glycolytic phenotype. This phenotype is characterized by hyperpolarized mitochondria, lower respiratory activity and supported by a lower intracellular environment. Moreover, A549 cells showed their membrane lipids with superior abundance of monounsaturated fatty acids and higher omega-6/omega-3 ratio. At last, when the cells are no longer bound to the extracellular matrix, mimic by trypsinization process, their mitochondria becomes more hyperpolarized, ROS production increases and their intracellular environment becomes more oxidized, increasing their tumour aggressiveness.