The human fibrosarcoma cell line HT-1080 was used as the negative control for E-cadherin expression. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HSC-2, HSC-3, HSC-4, KB, and FaDu), or a mixture of DMEM and Ham’s F-12 (SAS), or minimal essential medium (HT-1080), supplemented with 10% fetal bovine serum (FBS) in a humidified incubator (37°C, 5% CO2). Inhibition of Cox2 using its specific inhibitors HSC-2 and HSC-4 cells were seeded in six-well plates at a density of 2 × 105 cells per well and incubated overnight in 10% FBS medium. The

cells were then treated with different selective Cox-2 inhibitors: 50 μM of celecoxib (Toronto Research Chemicals, Toronto, Ontario, Canada), 80 μM of NS-398 (Cayman Chemical, Ann Arbor, MI, USA), or 20 μM of SC-791 (Calbiochem, Selleck NU7441 Darmstadt, Germany). These concentrations of each Cox-2 inhibitor were

found to be optimal with no toxic effect on cell viability up to 48 h based on our preliminary experiments for this purpose. Treatments with only dimethyl sulfoxide (DMSO) (Nacalai Tesque, Kyoto, Japan) used as a solvent for the inhibitors were set as the control. For the evaluation of changes in gene expression associated with Cox-2 inhibition, total RNA was extracted after a 12-h incubation. Quantitative real-time PCR BAY 57-1293 mw Total RNA from cell lines or fresh frozen tissues was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) and reverse-transcribed into cDNA using random hexamer primer and SuperScript II reverse transcriptase (Invitrogen)

according to the manufacturer’s Low-density-lipoprotein receptor kinase instructions. Quantitative real-time polymerase chain reaction (PCR) was performed using the 7500 Fast Real-Time PCR system instrument and software (Applied Biosystems, Foster City, CA) following the manufacturer’s protocol. Specific primers and probes were obtained from Applied Biosystems as TaqMan® Gene Expression Assays, with the following IDs: human E-cadherin/CDH-1, Hs00170423_m1; Snail/SNAI1, Hs00195591_m1; SIP1/ZFHX1B, Hs00207691_m1; twist/TWIST1, Hs00361186_m1; Cox-2/PTGS2, Hs01573471_m1; and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)/GAPDH, Hs99999905_m1. The PCR amplification conditions were: 20 s at 95°C followed by 40 cycles of 3 s denaturation at 95°C and 30 s annealing at 60°C. We quantified the relative expression levels of the genes by the standard curve method, and we compared the levels after normalization using those of GAPDH used as an endogenous control. Flowcytometric analysis For the quantitative analysis of E-cadherin expression at protein level, we harvested cells that had been treated with each of the selective Cox-2 inhibitors for 24 h, using a cell dissociation solution (C 5914, Sigma-Aldrich, St. Louis, MO).