Raloxifene

Human BT-474, MCF-7, and SKBR3 breast cancer cell lines were obtained from ATCC and were used in these studies because of differences in their expression of HER2 and ERα. BT-474 cells are HER2-negative and ERα-positive, MCF-7 cells are ERα-positive and HER2-negative, and SKBR3 cells are HER2-positive and ERα-negative (Neve et al. 2006 (link)). Cells were grown in phenol red–free Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 10 μg/mL streptomycin (Life Technologies) under 5% CO₂ at 37°C. Three days before treatment, the cells were incubated with DMEM/F12 supplemented with 10% charcoal-dextran–stripped FBS (Gemini Bio-Products). Recombinant human heregulin-β1 (HRG) was purchased from Leinco Technologies, Inc. and used at a final concentration of 20 ng/mL to activate the HER2 signaling pathway. Estradiol, raloxifene, tamoxifen, methylparaben (MP), ethylparaben (EP), propylparaben (PP), and butylparaben (BP) were purchased from Sigma-Aldrich Co. LLC. The estrogen receptor antagonist ICI 182,780 was purchased from Tocris Bioscience. The compounds were dissolved in ethanol. The final concentration of ethanol was 0.1%, which had no effect on the cells. An ethanol vehicle was used for the control cells.

MTT cell viability assay kit was purchased from Promoter Biotechnology Ltd. Cells were seeded in a 96-well plates (3,000 per well) in triplicates and then incubated with desired concentrations of Raloxifene (Sigma) at 37°C for 24 hours. MTT (10 μL) was added to each sample and incubated for 4 h. Then, 100 μL N, N-dimethylformamide solubilization solution was added to each well and incubated for 4 h and the absorbance was read at 470 nm.

The human PDAC cell lines L3.6pl and AsPC-1 were used for in vitro and in vivo studies. AsPC-1 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). L3.6pl is a secondary highly metastatic human pancreatic adenocarcinoma cell line derived from an orthotopic mouse xenograft model [15 ]. All cell lines were maintained in Dulbecco’s Minimal Essential Medium (Invitrogen GmbH, Karlsruhe, Germany), supplemented with 10% fetal bovine serum and 1% penicillin streptomycin and were incubated in a humidified atmosphere of 5% CO₂ at 37 °C.
Raloxifene was purchased from Sigma-Aldrich (Schnelldorf, Germany) and was dissolved in 0.1% dimethyl sulfoxide (DMSO, Carl Roth GmbH & Co. KG, Karlsruhe, Germany). IL-6 was purchased from Invitrogen GmbH (Karlsruhe, Germany) and was dissolved in acetic acid. Anti-STAT3, anti-phosphorylated-STAT3 (Y705) and anti-gp130 antibodies were obtained from Cell Signaling Technology (Frankfurt am Main, Germany).