Figure 3

Benzylguanine inhibits tamoxifen-resistant breast cancer growth, induces cell cycle arrest and apoptosis and sensitizes resistant breast cancer cells to antiestrogen therapy Tamoxifen-resistant MCF7 breast cancer cells were treated with different concentrations of BG and ATP growth inhibition assay was performed after 48 h and 72 h . (A) BG led to significant growth inhibition in a dose-dependent manner (BG IC₅₀ = 140 µmol/L, at 48 h). (B) Tamoxifen-resistant MCF7 cells were treated with BG for 24 h and TAM or ICI was added for another 48 h before growth inhibition ATP assay was performed. BG had a direct inhibitory effect and further enhanced the activity of TAM/ICI. (C) Tamoxifen-resistant MCF7 breast cancer cells were treated in the presence or absence of BG (IC₅₀ = 140 µmol/L) and 24 h later TAM (1 µmol/L) or ICI (1 µmol/L) was added for 48 h. p53 expression was not changed significantly, MGMT was decreased significantly by 90% in the BG- and BG + TAM/ICI-treated cells. ERα expression was increased by TAM and decreased by BG or BG + TAM/ICI. (Control (□); 4-OH TAM ; ICI ; BG ; 4-OH TAM + BG ; ICI + BG ). (D) Tamoxifen-resistant MCF7 cells were treated with different concentrations of BG and MGMT, p21, cytochrome C expression was determined by Western blot analysis. BG in a dose-dependent manner led to induction of p21 expression, release of cytochrome C and inhibition of MGMT. (Control (□); BG 40 µmol/L ; BG 60 µmol/L ;BG 80 µmol/L ;BG 100 µmol/L ;BG 120 µmol/L ;BG 140 µmol/L ;BG 160 µmol/L ). (E) Cytochrome C PUMA and p21 correlative expression was determined by Western blot analysis in BG (140 µmol/L) and BG combination therapy showing increased release of apoptosis mediators in the BG and BG combination therapy conditions. (Control (□); 4-OH TAM ; ICI ; BG ; 4-OHTAM + BG ; ICI + BG ). (F) Tamoxifen-resistant MCF7 cells were treated with BG (140 µmol/L) for 24 h before either TAM or ICI was added, for another 48 h before cells were harvested and cleaved PARP expression was measured by Western blot. Cleaved PARP was increased by single agents (TAM or ICI or BG) compared with control and it was increased further by combination therapy (TAM + BG/ICI + BG). (Control (□); 4-OH TAM ; ICI ; BG ; 4-OHTAM + BG ; ICI + BG ).