Estrogen Receptor-Reporter Activity Assay to Screen Phytoestrogen Estrogenic Activity
Transcrição
Estrogens are steroid hormones that act as endocrine signaling molecules and bind to nuclear receptors — estrogen receptors beta, forming estrogen-beta receptor complexes.
These ligand-receptor complexes dimerize and form homodimers in the cell cytoplasm. The resultant homodimer enters the nucleus, binds to the estrogen response element, ERE — a specific DNA sequence within the target gene's promoter — and initiates transcription.
To determine the estrogenic activity of phytoestrogens — plant-derived secondary metabolites that mimic estrogen function — begin with a multi-well plate containing reporter cells suspended in an appropriate medium.
The reporter cells are engineered to overexpress the estrogen receptors beta and transfected with a luciferase gene fused to ERE promoter. Add the sample solution containing phytoestrogens and incubate.
During incubation, the phytoestrogen binds to the estrogen receptors, beta — expressed in the reporter cells. Once bound, the phytoestrogen-receptor complexes dimerize and translocate into the nucleus.
Inside the nucleus, the dimerized complex binds to the ERE. The binding initiates luciferase gene transcription, producing the luciferase enzyme. Next, remove the medium and add a reagent containing a substrate for luciferase. Incubate to allow the expressed luciferase enzyme to oxidize its substrate and produce luminescence.
Measure the luminescence, which confirms the estrogenic activity of the compound in the sample.
