Cell proliferation assay

An induction of the ER pathway is commonly linked with increased proliferation of estrogen-sensitive cells. Thus, a standard assay for assessing the estrogenic potency of a compound is the so-called “E-screen assay” that uses the MCF-7 cell line and was established by Soto et al. (23). We adapted this assay for the use of Ishikawa cells. 600 cells/well were seeded in 96-well plates and allowed to attach for 24 h. Afterwards, they were incubated with the test compounds diluted in 100 μl of DMEM/F-12 medium, containing 5% CD-FCS, 1% penicillin/streptomycin and a final solvent concentration of 0.15% (v/v) DMSO in triplicates. Incubation solutions were renewed after 3 days to ensure the sufficiency of nutrients in the media. After a total of 6 days of incubation, cells were fixed by addition of 10 μl/well of a trichloroacetic acid solution (50% m/v). After 1 h of storing at 4°C, the plates were washed four times with water and were allowed to dry overnight. Subsequently, wells were incubated with a 0.4% (w/v) solution of sulforhodamine B (SRB) at room temperature for 1 h. After four times washing with 1% (v/v) acetic acid, wells were allowed to dry overnight. Finally, 100 μl/well of Tris buffer (pH 10) was added to resolve protein-bound SRB and the absorbance of the wells' content was measured at 570 nm with a PerkinElmer Victor³V plate reader. All measured values were related to the respective solvent control [0.1–0.2% (v/v) DMSO].