Quantifying the Effects of Common in vitro Cell Culture Techniques on Glutathione Depletion and Cellular Viability via Breast Cancer Cells and Reactive Oxygen Species (ROS)

Abstract

Glutathione (GSH), an important antioxidant, is essential for proper mammalian biochemical function. Its mechanism as an antioxidant involves the neutralization of reactive oxygen species (ROS) to less harmful compounds like water. ROS are neutralized using oxidation/reduction reactions, where glutathione reacts with a ROS forming oxidized glutathione (GSSG) and a neutral species, like water, in the process. GSSG is then reduced to GSH by glutathione reductase. Therefore, a proper in vivo concentration of GSH may determine a cell’s ability to survive stressful conditions, such as the presence of drug compounds: benserazide, hydrogen peroxide, or buthionine sulfoximine (BSO). The research presented in this thesis shows that common cell culture techniques, such as media replacement and cell washing, causes cells to be more susceptible to these drugs. To analyze the effects of the drugs, IC50 curves were constructed with results from cell viability assays under three cell treatment conditions: no media replacement, media replacement, and two times wash with PBS followed by media replacement. These experiments were performed using two triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468. One suspected cause of increased susceptibility is loss of total GSH inside of the cells and to measure total GSH, a luminescent assay was used. However, we concluded from experimentation that an increase in cell susceptibility to drugs is not due to loss of GSH. Future research is needed to determine the cause. Finally, these results suggest that procedures used in in vitro cell culture studies can cause changes to the cell viability and that these results should urge researchers to develop a more universal cell culture protocol that is comparable to in vivo conditions.