NS398 was reported to cause significant growth inhibition in HCA-7 colon carcinoma cells (Zhang and DuBois, 2001). www.selleckchem.com/products/Gefitinib.html It inhibits PGE2 synthesis and arrests cell cycle in G1 phase by enhancing p27KIP1 expression (Hung et al, 2000). NS398-dependent apoptosis in colon cancer cells occurs through a cytochrome c-dependent pathway (Li et al, 2001). Reducing VEGF levels with NS398 treatment refers to its anti-angiogenic effect (Abdelrahim and Safe, 2005; Huang et al, 2005). Inhibitory effects of NS398 on cancer invasiveness and metastatic growth have been proven both in vitro in cell culture (Abiru et al, 2002; Yao et al, 2004; Chen et al, 2006; Banu et al, 2007; Leung et al, 2008) and in vivo in animal model experiments (Chen et al, 2006; Leung et al, 2008).

Therapeutic effects of NS398 can be exerted by downregulation of matrix metalloproteinase-2 expression (Yao et al, 2004; Leung et al, 2008), blocking of epidermal growth factor receptor transactivation (Banu et al, 2007) or inhibition of HGF-induced invasiveness (Abiru et al, 2002; Chen et al, 2006). However, the complete molecular background of NS398 treatment on colon adenocarcinoma cells has not been analysed yet. The aims of this study were to analyse the gene expression modulating effect of NS398 selective COX2 inhibitor on the HT29 colon adenocarcinoma cell line and to correlate this effect to the modulation in gene expression observed during normal-adenoma and normal-CRC transition when biopsy samples were analysed. Materials and methods Cell culture HT29 colon adenocarcinoma cells were cultured at 37��C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, St Louis, MO, USA) containing gentamycin and 10% FCS.

In six-well plates, 300000cells per well were cultured for 1 day, and were then treated with 10, 25 and 100��M NS398 (Sigma-Aldrich, diluted in DMSO) for 72h in FCS-free medium. 0.1% DMSO was used as control. Total RNA was extracted from three samples treated with 100��M NS398 and from three untreated controls for microarray analysis. In parallel, 40000cells Entinostat per slide were cytocentrifuged and fixed for immunocytochemical analysis. MTT cell proliferation assay In 96-well plates, 5000 HT29 cells per well were maintained for 24h in 100��l RPMI-1640 medium containing 10% FCS, after which, the cells were treated with 10, 25 and 100��M NS398 (Sigma-Aldrich, diluted in DMSO) for 48 or 72h in FCS-free medium. A volume of 0.5mgml?1 of MTT (methylthiazolyldiphenyl-tetrazolium bromide, Sigma-Aldrich) was then added to each well, and the cells were incubated for 4h at 37��C.