Gefitinib iressa zd1839

Human TNBC cell lines, and the murine 4T1 mammary carcinoma cell line, were obtained from ATCC, Manassas, VA, USA and maintained in RPMI 1640 medium containing 5% FBS and 10 μg/mL bovine insulin under standard conditions. Identity of Hs578T cells, which were obtained from ATCC in 2001, was confirmed by short-tandem repeat profiling by CellBank Australia (Westmead, NSW, Australia) in December 2012. Cryopreserved stocks of other cell lines (purchased in 2010 from ATCC) were established within 1 month of receipt, and fresh cultures for use in experiments were established from these stocks every 2-3 months. All cell lines tested negative for mycoplasma contamination. Stable downregulation of IGFBP-3 was achieved by transfecting 2 × 10⁶ cells with SureSilencing shRNA or negative control plasmids according to the manufacturer’s instructions (Qiagen, Melbourne, VIC, Australia) and selection using hygromycin B. Cell lysates and media were collected post-selection to validate knockdown efficiency by qRT-PCR, radioimmunoassay and immunoblotting as previously described [13 (link)].
Gefitinib (Iressa; ZD1839, AstraZeneca) and FTY720 (Fingolimod; Gilenya, Novartis) were obtained from MedChem Express, Monmouth Junction, NJ, USA. Desmethyl erlotinib (OSI-420) was obtained from AdooQ BioScience, Irvine, CA, USA.

Estradiol (E₂), 4-hydroxytamoxifen and calcipotriol (MC 903) were purchased from Sigma (St. Louis, MO, USA). Cell culture medium was obtained from Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Hyclone Laboratories Inc. (Logan, UT, USA) and the antiestrogen ICI-182,780 (Fulvestrant) from Zeneca Pharmaceuticals (Wilmington, DE, USA). Gefitinib (Iressa, ZD1839) was kindly provided by AstraZeneca (Wilmington, DE, USA). U0126 was from Millipore (MA, USA). Trizol and the oligonucleotides for real time polymerase chain reaction (qPCR) were from Invitrogen (CA, USA). The TaqMan Master reaction, probes, capillaries, reverse transcription (RT) system and the cell proliferation assay (XTT) were purchased from Roche (Roche Applied Science, IN, USA). MCF-7 nuclear extract was purchased from Santa Cruz Biotechnology Inc., (CA, USA). The VDR antagonist (23S)-25-dehydro-1-hydroxyvitamin D3-26,23-lactone (TEI-9647) and 1α,25-dihydroxycholecalciferol (calcitriol) were kindly donated from Teijin Pharma Limited (Tokyo, Japan) and Hoffmann-La Roche Ltd. (Basel, Switzerland), respectively.