Figure 4
Comparison of pyranocoumarins with antiestrogens Faslodex™ and 4-hydroxytamoxifen. (a), (b) Effects of decursin (D) and decursinol angelate (DA) versus decursinol (DL) on estrogen-stimulated growth of MCF-7 cells as sparsely seeded colonies. MCF-7 cells (~10% confluent) were seeded in six-well plates in phenol-red free improved minimum essential medium (PRF-IMEM) with 5% charcoal-stripped serum without insulin with solvent, a pyranocoumarin or a known antiestrogen for 3 days. Without medium change, 0.1 nM 17β-estradiol (E2) was added for another 9 days. After fixation with 1% glutaraldehyde, the cells remaining attached were stained in situ with 0.02% crystal violet and photographed. (a) Image of representative wells. (b) Cell number estimated by dissolving the crystal violet in 70% ethanol (4 ml/well) and then optical density (OD) values measured at 570 nm with the reference filter of 405 nm. Results (mean ± standard error) represent triplicate wells. Analysis of variance (ANOVA): D at concentrations 10–50 μM (D10-D50) versus (+)E2, P < 0.0001; DA10-DA50 versus (+E2), P < 0.0001; DL10-DL100 versus (+)E2, P > 0.5; Faslodex™ versus (+)E2, P < 0.0001; 4-hydroxytamoxifen (4-HT) versus (+)E2, P < 0.001. (c) Comparison of effects of D with those of Faslodex™ and 4-HT on ERα and cleaved poly(ADP-ribose) polymerase (c-PARP) in MCF-7 cells in the hormone deprivation/restimulation model. Fresh medium with 1 nM E2 without or with D, or with known antiestrogen was replenished to respective flasks for 24 hours and the cellular extract analyzed by western blot. (d) Effect of combining D and Faslodex™ on basal and estrogen-stimulated colony growth in PRF-IMEM in six-well plates. Various treatments started at seeding for 5 days. Each bar represents mean ± standard error of the mean for triplicate wells. Results for one of two experiments, with identical patterns of responses. Concentrations: D, 10 μM; Faslodex™, 0.1 nM; estradiol, 0.1 nM. Columns sharing the same letter are not different at P = 0.05. (e) Effect of D on colony growth of MCF-7 cells stimulated by increasing levels of estradiolin six-well plates. Various treatments started at seeding for 5 days. Each bar represents mean ± standard error of the mean for three independent experiments, each with duplicate or triplicate wells. D concentration, 10 μM. ANOVA: E2 effect, P < 0.001; D effect, P < 0.001; E2 × D interaction, P > 0.5.