Figure 5
Effects of pyranocoumarins on estrogen receptor alpha protein stability and mRNA level in MCF-7 cells. (a) Effect of decursin (D) and decursinol angelate (DA) on estrogen receptor (ER)α protein abundance in the absence and presence of estrogen. MCF-7 cells were cultured in phenol-red free improved minimum essential medium (PRF-IMEM) with 5% charcoal-stripped FBS (CSS) and without insulin for estrogen starvation for 3 days. For estrogen stimulation, D or DA was added 2 hours before 1 nM 17β-estradiol (E2) was added without medium change. Cell lysates were prepared 48 hours after D or DA treatment for western blot analyses. In the time course, D and E2 treatments initiated simultaneously. c-PARP, poly(ADP-ribose) polymerase. (b) Effects of D (50 μM) on ERα protein stability in MCF-7 cells treated in complete medium with cycloheximide (CHX, 50 μg/ml) for 15, 24, 35, and 48 hours. Cell lysates analyzed by western blot for ERα and the short-lived cyclin D1 to establish potency of CHX. (c) Effects of D, DA and decursinol (DL) on the steady-state level of mRNA transcripts of ERα and the estrogen-responsive gene pS2 detected by RT-PCR. MCF-7 cells were cultured in PRF-IMEM with 5% CSS and without insulin for estrogen starvation as above. D, DA, DL or Faslodex™ was added 2 hours before 1 nM E2 without medium change. After 24 hours, cells were harvested for RNA isolation and RT-PCR. The housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene serves as a loading control. (d) Acute time-course effect of decursin on ERα and estrogen-responsive genes pS2 and cyclin D1 detected by RT-PCR. D and E2 treatments initiated simultaneously.