Characterizing Metabolic Changes in Cancer Cells after Treatment with an Extract Created from Walnuts

Abstract

One of the main hallmarks of cancer cells is the reprogramming of energy metabolism to support the high energy and metabolite demands of constant proliferation. Although previously observed as mainly a shift from oxidative phosphorylation to glycolysis, also known as the Warburg Effect, recent research has shown that cancer cells can utilize both pathways to make energy, which has been referred to as metabolic flux. This suggests that cancer cells can adapt to their environment by reactivating or continuing to use the oxidative phosphorylation pathway to make energy. Thus, the energy metabolism of cancer cells is of therapeutic interest. The triple‐negative breast cancer cell line MDA‐MB‐231 can undergo metabolic flux, making it an ideal cell line to study the effects of metabolism‐altering drugs. Previous work from our lab showed that treatment with a Walnut Extract (WE) induced cell death in MDA‐MB‐231 cells, as well as HeLa cells. We also observed that the WE treatment targets the mitochondria by destabilizing the mitochondrial outer membrane potential, suggesting that the extract may be able to impact oxidative phosphorylation. To examine the effects on energy production, we have taken advantage of the Agilent Seahorse XFe96 Cell Metabolic Analyzer to monitor, in real time, metabolic changes in MDA‐MB‐231 and HeLa cells treated with WE. WE treatment had impacts on both glycolytic and oxidative phosphorylation processes in both cell lines. Both time‐ and dose‐dependent responses were observed, with WE treated MDA‐MB‐231 cells exhibiting a 70% reduction in basal oxygen consumption rate compared to vehicle treated cells. Additionally, basal glycolytic rate was reduced by up to 41%, and compensatory glycolysis was reduced 70%. In HeLa cells, basal oxygen consumption rate was reduced by 74%, with a 56% reduction in basal glycolytic rate and a 76% reduction in compensatory glycolysis. Quantification of ATP production rate showed an overall reduction of 73%, with a 91% reduction in mitochondrially contributed ATP production, as well as a 47% reduction in glycolytic ATP production in MDA‐MB‐231 cells. Quantitation of total ATP within the cell using a separate assay shows an 62% decrease in ATP in treated HeLa cells. We have also looked at the status of mTOR and AMPK in the cells after treatment with the WE, because of their role in energy sensing and energy production. Interestingly, our preliminary data suggests that phosphorylated mTOR levels do not change and we also do not detect any phosphorylation of AMPK, suggesting that the WE is not effecting its changes on these proteins despite the reduction in overall ATP in the cell. Taken together, our results suggest that the WE treatment can alter and reduce the rates of glycolysis and oxidative phosphorylation, and by extension, the metabolic flux of these cancer cells.