25 Evaluating the Lipotoxicity of Various Dietary Fatty Acids on Breast Cancer Cells

Faculty Mentor: Amandine Chaix (Nutrition and Integrative Physiology, University of Utah)

Introduction

As of 2020, breast cancer is the most commonly diagnosed cancer worldwide, accounting for 11.7% of all cancer diagnoses. In women, breast cancer is the leading cause of cancer related death, and the incidence of breast cancer is rising. Obesity is one factor contributing to this increased prevalence.^[1] Fatty acids are essential for cellular function and survival. However, the accumulation of excessive fatty acids, particularly in non-adipose tissue as observed with obesity, can lead to lipotoxicity, a condition characterized by cellular damage and ultimately cell death.^[2][3] Lipotoxicity induced cell death occurs through various mechanisms. An overabundance of saturated fatty acid (SFA), such as palmitic acid, can induce apoptosis while polyunsaturated fatty acid (PUFA) can cause ferroptosis. Notably, an overabundance of has been shown to induce apoptosis while. Conversely, monounsaturated fatty acid (MUFA), such as oleic acid, can be protective against the toxicity of both SFA and PUFA.^[4]

In this study, we use E0771 cells, a murine of breast cancer cell line, to investigate the lipotoxic effects of common dietary fatty acids: palmitic acid (SFA) and oleic acid (MUFA). We test the hypothesis that fatty acid structure and saturation correlates with its toxic effects at high concentrations. Specifically, we determine the LD50 of palmitic acid by dose response curve analysis. This research aims to clarify how dietary fatty acid composition may influence breast cancer cell survival and proliferation.

Methods

Cell growth assay: E0771 cells were seeded into 96-well plates at 200,000 cells/mL and cell number assessed after 0, 48, 72, and 96 hours of growth. Cell number was evaluated using PrestoBlue, a fluorescent assay that quantifies cellular metabolic activity as a proxy of cell number. The fluorescence was measured using a Varioskan LUX microplate reader with SkanIt Software.

Effect of fatty acid treatment: Cell growth assays were conducted to evaluate the effects of palmitic acid (SFA) and oleic acid (MUFA). Fatty acid stock solutions were DMSO as the vehicle and cells treated with 12.5 µM, 25 µM, 50 µM, and 100 µM of each fatty acid. Media only and 1% DMSO treated wells served as controls. Four replicates were completed for each condition. To determine the LD50 of palmitate, a dose-response curve was produced from the results of 72h growth in presence of the indicated concentrations of palmitate. To assess the protective effect of oleic acid, cells were co-treated with 50 µM oleic acid and 50 µM palmitic acid. Co-treatment was compared to cells treated with oleic acid only, palmitic acid only, media only, and 1% DMSO. PrestoBlue assays were performed at 48 and 72 hours to assess the effects of co-treatment over time.

Results

Effect of palmitic acid on E0771 growth

When E0771 cells were grown in presence of palmitic acid at concentrations of 50 µM and 100 µM, the fluorescence, measured in arbitrary units (A.U.), decreased over the 96-hour growth period, indicating reduced cell viability. This result suggests that palmitate at concentrations higher than 50 µM is toxic to E0771 cells. Cells that were grown with 12.5 µM palmitic acid demonstrated more growth than both the media only and 1% DMSO controls. Cells treated with 25 µM palmitic acid also supported cell growth, though to a lesser extent than the 12.5 µM treatment and both controls (Figure 1). Based on the 72 hours growth curve, the calculated LD50 for palmitic acid was 22.26 µM (Figure 2).

Effect of Oleic acid on E0771 growth

As the concentration of oleic acid treatment increased, E0771cell growth decreased. However, oleic acid did not exhibit lipotoxic effects at concentrations up to 100 µM at which growth was only reduced by half (Figure 3).

Effect of palmitic plus oleic acid on E0771 growth

Co-treatment with the 50 µM oleic acid and 50 µM palmitic acid resulted in cell growth comparable to that of media only and 1% DMSO controls. At both 48 and 72 hours, co-treated cells demonstrated higher viability than those treated with either 50 µM oleic acid or 50 µM palmitic acid alone. Among treatments of a single fatty acid, oleic acid consistently supported more cell growth than palmitic acid (Figure 4).

Discussion

Our results show that, at similar concentration, palmitic acid, a SFA, exhibited toxic effects, whereas oleic acid, a MUFA, demonstrated no toxic effect. These results suggest a correlation between fatty acid structure and its effect on E0771 cells at high concentrations. We also observed a protective effect of oleate co-treatment against palmitate toxicity, supporting the hypothesis that MUFA exhibits protective effects against lipotoxicity induced from excessive SFA in E0771 cells.

The PrestoBlue cell growth assay does not allow us to distinguish between cell death and cell growth arrest. Future analysis to quantify apoptosis, ferroptosis, and cell growth will clarify by which mechanisms individual and combined free fatty acids (FFA) regulate cell growth. For example, we could quantify apoptosis through monitoring caspase activity, ferroptosis through monitoring 4 HNE, and cell cycle through monitoring CSE (carboxyfluorescein succinimidyl ester) dilution by flow cytometry. The effect of FFAs on ferroptosis requires further investigations. Treatment with a common PUFA, such as linoleic acid, followed by co-treatment with a MUFA to evaluate possible protective effects in E0771 cells, will be performed in future studies.

All experiments were conducted using a high glucose media (4.5 g/L +5% FBS). While a high glucose media is typically used for in vitro experiments, it has been shown to alter proliferation of various cells.^[5] Physiological glucose levels are lower, typically ranging from 0.8-1.4 g/L.^[6] It remains unclear whether there is a significant difference in the cell growth of E0771 cells in a low glucose media (1 g/L + 5% FBS) versus a high glucose media. Future studies may be conducted to replicate these experiments using low glucose media to determine whether glucose concentration significantly alters the observed effects. This is important for future translational applications of our findings.

Bibliography

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