The expression of genes involved in selleck chemicals cell proliferation and cell cycle regulation (such as overexpressed CDKN3, BTG2, TGFB1, CNOT8, KAT2B, RARRES3, CDKN2C, RARRES1, MAGED1, PPAP2A, MXD4, TENC1, SESN1, and downregulated CDCA4, VEGFA), intracellular signal transduction, transcription regulation, metabolic and transport processes and apoptosis (overexpressed CDKN2C, BIK, CASP6, TIA, DAPK3, and dowregulated ANXA1, CEBPB, CBX4) are mainly changed under NS398 treatment. However, the function of several differentially expressed transcripts is not known yet. The functional classification of genes is represented in Figure 1. Figure 1 Functional classification of differentially expressed genes in HT29 cells under NS398 treatment. (A) Distribution of differentially expressed transcripts in the main cell functional groups.

(B) Distribution of downregulated transcripts in the main cell … In correlation with the mRNA expression findings, significant dose-dependent cell proliferation inhibition was measured using MTT assay, which was carried out to optimise the treatment concentration of NS398 COX2 inhibitor. Changes in colorectal adenoma and cancer-related mRNA expression patterns under NS398 treatment Seventeen of these 20 genes changed in a reverse manner in HT29 colon adenocarcinoma cells under NS398 COX2 inhibitor treatment, 14 of them (including upregulated somatostatin, claudin 8, peptide YY, and downregulated cadherin 3, KIAA1199) at a significance of P<0.05 (Figure 2A). The expression of 12 of the 38 CRC-related markers (such as carbonic anhydrase 7, interleukin 8, melanoma cell adhesion molecule) was changed in a reverse manner under NS398 treatment (Figure 2B).

Figure 2 Changes in colorectal adenoma and cancer-related mRNA expression patterns under NS398 treatment. (A) Expression of adenoma vs normal discriminatory genes in biopsy samples and in HT29 colon adenocarcinoma cells under NS398 treatment (B). Expression of … HT29 immunocytochemistry and western blot results Dose-dependent inhibition of COX2 protein expression was observed under NS398 treatment. COX2-positive cell/total cell ratio was 80.5% in untreated control samples, whereas it decreased to 77.0% under 10��M, to 61.2% under 25��M NS398 treatment. Further elevation of the NS398 dose (100��M) caused a significant decrease in the positive cell ratio (53.1%).

Strong granular and/or diffuse cytoplasmatic immunostaining was detected in COX2-positive AV-951 cells (Figure 3A and B). Western blot results showed correlation to the immunocytochemistry findings (Figure 3C). More considerable reduction in COX2 protein expression was detected after 96h of NS398 treatment at 50 and 100��M concentrations. Figure 3 The decrease in COX2 protein expression under NS398 treatment. Dose-dependent inhibition of COX2 protein expression was observed under NS398 treatment. Strong granular and/or diffuse cytoplasmatic immunostaining was detected in COX2-positive cells. ( …