Figure 3
FR58P1a is a protonophore uncoupler of OXPHOS in mitochondria isolated from TA3/Ha cancer cells. (a) Effect of predicted inactive compounds and FR58P1a on ATP levels in glycolytic (red points) and oxidative (blue points) subpopulations of MDA-MB-231 breast cancer cells at 4 h of exposure. (b,c) Effect of FR58H8 and FR58P1a on complex I-dependent respiration (stimulated with glutamate plus malate) in state 4o and 3 u, in presence of 1 µM oligomycin and 0.2 µM CCCP, respectively. (d) Effect of bromohexanoic acid (BHA), FR58H8 and FR58P1a on Respiratory Control Ratio (RCR) in presence of glutamate plus malate (G + M), which was calculated as the respiration in state 3ADP divided by that in state 4. (e) Double logarithmic plots of uncoupling effect of both regioisomers FR58P1a and P1b in mitochondria isolated from TA3/Ha cells. In blue circles, the effect of FR58P1a and FR58P1b on mitochondrial respiration in state 4o (1 µM oligomycin addition); in red circles, effect of compounds on maximum electron flux (state 3 u by 0.2 µM CCCP addition). Lines were fitted by regression of log-log plot, obtaining the slope log-log plot and K0.5 values. (f) Dependence of electron transport chain, mPTP, ANT, FoF1-ATP synthase and (g) membrane fluidity in the uncoupler effect of FR58P1a. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, vs. Control (DMSO). n.s. not significant.
