Figure 3: DDA is the most potent natural inhibitor of ChEH.
MCF-7 cells were incubated with 0.6 μmol l−1 [14C]-5,6α-EC at 37 °C for 8 h with or without the indicated concentrations of DDA or C17. ChEH activity was assayed by measuring the conversion of [14C]-5,6α-EC to [14C]-CT by thin-layer chromatography (TLC) and quantified by densitometric analysis. (a) On the left panel, a representative autoradiogram of a TLC run from three independent experiments; on the right panel, the dose–response curves of the inhibition of [14C]-CT formation measured by TLC with DDA (circle) or C17 (square) at the indicated concentrations. The curves represent the mean±s.e.m. of three independent experiments and were used to calculate the IC50. (b) Modality of ChEH inhibition by DDA. The relationship between the conversion rates of 5,6α-EC to CT and DDA concentrations is shown using 0.5, 1 and 5 μM DDA with MCF-7 cell extracts. A double reciprocal plot of velocity versus [14C]-5,6α-EC at the given DDA concentrations is presented. Experiments were repeated at least three times in duplicates. The data presented are the mean±s.e.m. of all experiments. (c) Measurement of ER-dependent transcriptional activity in MELN cells. Cells were incubated with the solvent vehicle (control), 10 nM E2 or increasing concentrations of DDA with or without 10 nM 17β-estradiol (E2) or 1 μM Tam with 10 nM E2. Experiments were repeated at least three times in duplicates. The data presented are the means±s.e.m. of all experiments. P<0.001 compared with control cells.
