4 androstenedione ad

From the ERα MCF-7-derived breast cancer cell line stably transfected with the human aromatase gene (MCF-7 control) as previously reported [37 (link)], different cell lines were established, namely: a tamoxifen-resistant cell line (Res-Tam), a fulvestrant-resistant cell line (Res-Fulv), an anastrozole-resistant cell line (Res-Ana) [38 (link)], and a letrozole-resistant cell line (Res-Let) [39 (link)]. The Res-Tam cell line was established by exposing the MCF-7 control cells during 25 weeks to increasing concentrations (1 and 3 μM) of 4-hydroxy-tamoxifen (OH-Tam, Sigma, St Louis, MO, USA) in Dulbecco’s Modified Eagle Medium without phenol red, supplemented with 3% steroid depleted, dextran-coated and charcoal-treated fetal calf serum (DCC medium) containing 25 nM 4-androstenedione (AD) (Sigma, Saint Louis, MO, USA). All of the human cell lines were maintained at 37 °C in the appropriate medium supplemented with 10% foetal calf serum.

A new letrozole-resistant cell line, denoted Res-Let, was established from the ER+ MCF-7-derived breast cancer cell line stably transfected with the human aromatase gene (MCF-7aro cells, kindly provided by Dr Shiuan Chen) [28 (link)]. The MCF-7aro cells were exposed during a 20-week period to increasing concentrations (1, 3 and 5 μM) of letrozole (Novartis, Basel, Switzerland) in Dulbecco’s modified Eagle’s medium without phenol red, supplemented with 3% steroid-depleted, dextran-coated, charcoal-treated fetal calf serum (DCC medium) containing 25 nM of 4-androstenedione (AD) (Sigma-Aldrich, St Louis, MO, USA). The previously described Res-Ana cells [6 (link)] were used as a model of acquired resistance to anastrozole. The cells were purged in DCC medium for 4 days prior to each experiment described below, then treated with 25 nM AD combined, or not, with the appropriate treatment. Media and treatments were changed every 2 days.