Combined HDAC and ER inhibition reduces proliferation and viability of tamoxifen-resistant cells. (A) Tamoxifen-resistant cells derived from MCF7 (TAMRM) cells were treated with and without 10 μM 4-hydroxy tamoxifen (Tam) and increasing concentrations of PCI-24781(PCI) for 72 hours and assayed for viability using a trypan blue dye exclusion assay. (B) TAMRM cells were treated with either vehicle (C), 200 nM PCI (P), 10 μM Tam (T) or the combination of PCI-24781 and 4-hydroxy tamoxifen (PT) for 72 hours and evaluated by colony formation assay. Colonies were counted after 2 weeks and considered formed if they contained approximately >100 cells. MCF7 and TAMRM cells were treated with vehicle (C), 200 nM PCI (P), 10 μM Tam (T) or the combination (PT) for (C) 24 hours and evaluated for cell cycle composition by flow cytometry or (D) 72 hours and evaluated for relative proliferation by MTS assay. (E) TAMRM cells were treated with either 0.5 mM valproic acid, 25 nM panobinostat, 1 μM vorinostat or 1 μM entinostat in presence and absence of 10 μM Tam for 72 hours and assayed for viable cells. (F) T47D and tamoxifen-resistant cells derived from T47D (TAMRT) cells were treated as in (D) and assayed for viable cells. Asterisk (*) indicates significant difference (P value <0.05) whereas (#) indicates insignificant difference (P value >0.05), compared to control group. HDAC, histone deacetylase.