The cell proliferation inhibitory effect of NS398 could be detected in our microarray analysis by causing cell cycle arrest in the G1 phase, as described earlier (Hung promotion et al, 2000). This can be mediated not only by p27KIP1 (Hung et al, 2000) but also by p18-INK4C (CDKN2C) and CIP2 (CDKN3) overexpression (the latter ones showed more than a 4.5-fold overexpression under NS398 treatment in our study). The p53-inducible gene BTG2 (we found to be 4.6-fold upregulated in HT29 cells after NS398 treatment) also contributes to the anti-proliferative activity of NS398 through its inhibition effect to G(1)�CS transition by reduction of cyclin D1 levels (Guardavaccaro et al, 2000). The cell proliferation inhibitory effect of NS398 has also been proven in MTT assay.

The inhibition of COX2 by NS398 results in the accumulation of arachidonic acid in cancer cells and, therefore, would trigger apoptosis, but the mechanisms by which NSAIDs induce cancer cells to apoptosis can also be COX2 independent. In this study, a wide range of pro-apoptotic genes in different phases of apoptosis were found to be overexpressed under NS398 treatment including TRAIL death ligand (TNFSF10), SIVA1 death receptor in CD27-induced pathway and molecules involved in the execution phase of apopotosis such as the APAF1 apoptosome protein and CASP6 effector caspase. Death-associated kinase-3 (DAPK3) inducing morphological changes in apoptosis was also upregulated by NS398 in HT29 cells. In accordance with the findings of Li et al (2001), NS398-dependent apoptosis in colon cancer cells occurred through a cytochrome c-dependent pathway in our experiments.

We found that the activation of the p53-dependent pathway can also trigger apoptotic processes via the cytochrome c pathway. Overexpression of tumour protein p53-inducible nuclear protein-1 and tumour protein p53-inducible protein-3 pro-apoptotic molecules indicates p53-dependent apoptosis. p73, which can transactivate p53-responsive genes causing cell cycle arrest and apoptosis, is also upregulated under NS398 COX2 inhibitor treatment. Celecoxib also caused overexpression of p73 tumour-suppressor gene in prostate cancer in a randomised controlled phase II pre-surgical trial (Sooriakumaran et al, 2009). Although only few genes involved in angiogenesis showed significant mRNA expression changes, in accordance with observations of Abdelrahim and Safe (2005) and Huang et al (2005), we also detected the downregulation of VEGF, one of the most important angiogenic Carfilzomib factors, besides the underexpression of others such as PTEN and IL18.