AGE Modulates Tamoxifen Response in Breast Cancer

Exogenous BSA-AGE and control BSA were produced as previously published [17 (link)]. For examination of AGE treatment, cells (400,000) were seeded into each well of a 6-well plate in serum-free media overnight before treatment with AGE metabolite (50ug/ml). Effects of AGE treatment on the phosphorylation of ERα, AKT, and ERK was assessed by Western blot as indicated below. Pharmacological targeting of the MAPK/ERK and PI3K/AKT pathways in the presence of AGE was achieved in serum-free media using the molecular inhibitors U0126 (ERK) and LY294002 (AKT) (Cell signaling Technology, Danvers, MA) at a concentration of 10uM for 12 h before exposure to AGE metabolites. To examine the effects of AGE metabolite on tamoxifen resistance, cells (3,000) were seeded into each well of a 96-well plate and were treated with the active metabolite of tamoxifen, 4-hydroxytamoxifen (0, 10 µM) (Sigma-Aldrich, St. Louis, MO) in combination with varying doses of AGEs (0, 5 µg/mL, 10 µg/mL, 50 µg/mL). After 24, 48, and 72 h, SRB staining was used to quantify cell growth as described previously [18 (link)].