Combined HDAC and ER inhibition reverses altered Bcl-2, c-Myc, and p21 expression. (A) Tamoxifen-resistant cells derived from MCF7 (TAMRM) cells were treated with increasing concentrations of PCI-24781 (PCI) for 24 hours and evaluated for estrogen receptor (ER), p21, c-Myc, Bcl-2 and GAPDH expression by western blot. (B) MCF7 and TAMRM cells were treated with vehicle (C), 200 nM PCI (P), 10 μM 4-hydroxy tamoxifen (Tam) (T), or the combination of PCI-24781 and 4-hydroxy tamoxifen (PT) for 48 hours and evaluated for ER, c-Myc, p21 and Bcl-2 expression by Western blot. (C) TAMRM cells were transfected with increasing concentrations of small interfering RNA (siRNA) for ESR1 and 24 hours later analyzed for BCL-2 expression. The blot depicts loss of ER protein with increasing concentrations of siRNA directed to ER. MCF7 and TAMRM cells were incubated in estrogen-free media for 48 hours and (D) treated with vehicle (DMSO) (C), 0.1 nM estradiol (E2) or 0.1 nM E2 + 10 μM Tam for 24 hours and (E) TAMRM cells were treated with increasing doses of E2 (0.1, 1 or 10 nM) in presence or absence of 200 nM PCI for 24 hours and relative BCL-2 mRNA expression was measured. (F) Relative BCL-2 mRNA expression was measured in complete media 24 hours post treatment with vehicle (C), 10 μM Tam, 200 nM PCI, 2 mM valproic acid or 20 nM panobinostat, in absence and presence of Tam. Asterisk (*) indicates significant difference (P value <0.05) whereas (#) indicates insignificant difference (P value >0.05), compared to control group. HDAC, histone deacetylase.