Figure 9

Inhibition of cellular bioenergetic parameters by YD0171 in HCT116 cells. Cells were treated with YD0171 (1 mmol/L) for 24 h, followed by extracellular flux analysis. (A) Left panel: mitochondrial function analysis. Four key parameters of mitochondrial function (basal respiration, ATP turnover, proton leak and maximal respiration) were assessed through the sequential use of 40 µmol/L oligomycin (ATP synthase inhibitor), 13.5 µmol/L FCCP (mitochondrial uncoupler) and 10 µmol/L rotenone + 1 µmol/L antimycin A (complex I and III inhibitors). The difference between the maximal and the basal respirations was considered the respiratory reserve capacity (the capacity of a cell to generate ATP via oxidative phosphorylation in response to increased demand for energy). Right panel: analysis of glycolytic function. A glycolytic stress test was used to estimate various parameters of cellular glycolysis (glycolysis, glycolytic capacity and glycolytic reserve), which was obtained with the sequential use of 25 mmol/L glucose, 5 µmol/L oligomycin (to block mitochondrial respiration and force the cells to rely on glycolysis for ATP production) and 100 mmol/L 2-deoxyglucose (2-DG, a glucose analog and inhibitor of glycolytic ATP production). Glycolytic reserve was calculated as the difference between the glycolytic capacity and glycolysis; this parameter is indicative of the cellular ability to increase the glycolytic rate upon increased energy demand. Acidification of carbon dioxide, the end product of the TCA cycle, which can be converted to bicarbonate, is considered a major contributor to nonglycolytic acidification. (B) Statistical analysis of the various bioenergetic parameters. Data shown as mean ± SEM; n = 6; *p < 0.05 and **p < 0.01 indicate significant inhibitory effects of YD0171 on the various bioenergetic parameters, compared with vehicle control.