Sk br 3

Four human breast cancer cell lines with different immunoprofiles, namely MDA-MB-361 (ER⁺ HER2⁺), MCF-7 (ER⁺ HER2^-), SK-BR-3 (ER^- HER2⁺), and MDA-MB-231 (ER^- HER2^-) were obtained from American Type Culture Collection (ATCC, USA) and were grown in culture flasks in HuMEC Basal Serum-free Medium (Thermo Fisher, USA) in order to prevent the influence of hormones and/or growth factors in regular serum-containing medium. For ER-positive cell lines (MDA-MB-361 and MCF-7), the cells were seeded in 96-well culture plates and incubated with various concentrations of AS extract, 17β-estradiol (0.1 µM, Sigma, USA), or the combination of 17β-estradiol (0.1 µM) and AS extract in medium for 48 h. For ER-negative cell lines (SK-BR-3 and MDA-MB-231), the cells were seeded in 96-well culture plates and treated with various concentrations of AS extract for 48 h. The cell viability and proliferative responses were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) cell proliferation enzyme-linked immunosorbent assay (ELISA) (Roche, USA), respectively.

HER2+ breast cancer cell line SK-BR-3 (WT) was obtained from the American Type Culture Collection and the cells were maintained in McCoy’s 5A medium (Hyclone, USA) containing 10% FBS (Hyclone, USA), 1% penicillin/streptomycin (Lonza, Switzerland), and 1% L-glutamine (Lonza, Switzerland). SK-BR-3 cells were cultured in 5% CO₂ at 37 °C. Trastuzumab resistant SK-BR-3 (TR) cells were additionally supplemented with 10μg/mL Trastuzumab (Herceptin), which was purchased from Roche (Basel, Switzerland).