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Glucocorticoid receptor (GR) antagonist activity, Progesterone receptor (PR) antagonist activity
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T47D alkaline phosphatase assay: T47D human breast cancer cells are plated in 96 well tissue culture plates at 104 cells per well in assay medium [RPMI medium without phenol red containing 5% (v/v) charcoal treated FBS and 1% (v/v) penicillin streptomycin]. Two days later, the medium is decanted and Mifepristone or control is added at a final concentration of 0.1% (v/v) dimethylsulfoxide in fresh assay medium. Twenty four hours later, an alkaline phosphatase assay is performed using a SEAP kit. The medium is decanted and the cells are fixed for 30 minutes at room temperature with 5% (v/v) formalin. The cells are washed once at room temperature with Hanks' buffered saline solution. Equal volumes (0.05 mL) of dilution buffer, assay buffer, and 1:20 substrate/enhancer mixture are then added. After 1 hour incubation in the dark at room temperature, the lysate is transferred to a white 96 well plate and luminescence is read using a LuminoSkan Ascen. A549 reporter assay: A549 human lung carcinoma cells are washed with Opti-MEM I. The medium is removed and lipid DNA complex solution (1.5 μg/mL of GRE luciferase reporter DNA in 8.5 mL?Opti-MEM I plus 6 μL/mL DMRIE-C reagent in 8.5 mL?Opti-MEM I, combined, mixed and incubated at room temperature for 40 minutes) is overlayed onto the cells in a T160 flask. The cells are incubated for 16 hours at 37℃ in a CO2 incubator. The DNA containing medium is removed and 30 mL of growth medium containing 5% (v/v) charcoal treated fetal bovine serum is added. After 5-6 hours, the cells are seeded in 96 well plates and incubated overnight at 37℃. Mifepristone is then added to each well followed by dexamethasone as a corticoid challenge. The cells are incubated for 24 hours. Luciferase assay buffer is added to each well and the cells are incubated for 30 minutes at room temperature. Luciferase activity is measured in a DYNEX Microlite plate on a TopCount.
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References:
1. Jiang, W., Allan, G., Fiordeliso, J. J., Linton, O., Tannenbaum, P., Xu, J., Zhu, P., Gunnet, J., Demarest, K., Lundeen, S. and Sui, Z. (2006) New progesterone receptor antagonists: phosphorus-containing 11beta-aryl-substituted steroids. Bioorg Med Chem. 14, 6726-6732
2. Ko, J. K., Huang, V. W., Li, R. H., Yeung, W. S., Ho, P. C. and Chiu, P. C. (2014) An in vitro study of the effect of mifepristone and ulipristal acetate on human sperm functions. Andrology. 2, 868-874
3. Goyeneche, A. A., Caron, R. W. and Telleria, C. M. (2007) Mifepristone inhibits ovarian cancer cell growth in vitro and in vivo. Clin Cancer Res. 13, 3370-3379
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