Atcc htb 22

Human cell lines (breast adenocarcinoma MCF-7 (ATCC ® HTB-22™)) and pancreatic carcinoma PANC1 (ATCC® CRL-1469™) were purchased from ATCC (Manassas, VA 20110 USA) collection. The cells were grown in 1×Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Rockville, MD, USA) conditioned medium, supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT, USA), and 5 μg/mL plasmocin™ (InvivoGen, San Diego, CA, USA) in a 5% CO₂ incubator at 37°C. At ~80% confluence, the cells were passaged at least five times before use in the experiments. Tamoxifen-resistant MCF-7 TMX and gemcitabine-resistant PANC1-GemR cells were cultured in media containing 10 μM tamoxifen and 0.01 μM gemcitabine, respectively, for over one year, authenticated in Szewczuk's lab.

The cytotoxicity of compounds 1, 3, 5 and 6 was measured through the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay^{33 (link)}, that is based in the conversion of the tetrazolium salt to a formazan product by viable cells. Two cell lines obtained from American Type Culture Collection (Manassas, Virginia, EUA) were used: HCT-116 (colorectal carcinoma, ATCC^® CCL-247™) and MCF-7 (breast adenocarcinoma, ATCC^® HTB-22). The assay was conducted essentially as described by Monteiro et al.^{34 (link)}. The HCT-116 and MCF-7 cells (1 × 10⁴ cells well⁻¹) were seeded in 96-well plates and, after 24 h, different concentrations (5 µM and 50 µM) of the compounds were added and incubated for 72 h. Doxorubicin (0.003 to 10 µM) and DMSO (0.5%) were used as a positive and negative controls, respectively. At the end of incubation, the supernatant was replaced with fresh medium containing MTT (0.5 mg/mL) for 3 h. The supernatant was, then, removed, and the MTT formazan product was dissolved in 150 μL DMSO. The absorbance was measured using a multiplate reader (Multiskan™ FC, Thermo Scientific, Finland) at 570 nm. All experiments were performed in duplicate. IC₅₀ values, along with 95% confidence intervals, were calculated by non-linear regression using GraphPad Prism 5.0 (Intuitive Software for Science, La Jolla, CA, USA).

The human brain endothelial cell line (hCMEC/D3) was obtained under licence from University Paris 05, CNRS, Institute Cochin, INSERM (Paris, France). The cell line was maintained and characterised in accordance to regularly updated protocols by Institute Cochin and certified mycoplasma free^{56 (link)}. Human breast cancer cell line (MCF-7) (ATCC^® HTB-22^™) and human mammary epithelial cell line (MCF-10A) (ATCC^® CRL-10317^™) were purchased from American Type Culture Collection (VA, USA). Endothelial Basal Medium (EBM-2) (#CC-3156) was from Lonza Group Ltd. (Basel, Switzerland). Dulbecco/Vogt Modified Eagle's Minimal Essential Medium (DMEM) (#D0819), basic fibroblast growth factor (#F0291), gelatin solution (#G1393), hydrocortisone (#H-0888), insulin (#I-1882) and phosphate buffered saline (PBS) (#D8537) were purchased from Sigma-Aldrich Co. LLC. (Seelze, Germany). Foetal bovine serum (FBS) (#16000044) and penicillin/streptomycin (#15140122) were obtained from Gibco-BRL, Carlsbad Life Technologies (CA, USA). Recombinant human EGF (AF-100-15) was from PeproTech EC Ltd. (Hamburg, Germany). HEPES (#S11-001) was from PAA Cell Culture Company (Pasching, Austria). The cell culture flasks and plates were purchased from Corning Inc. (NY, USA).