Down regulation of PGE₂ by celecoxib is not sufficient to inhibit cell growth or induce apoptosis Free

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Background: Several in vitro, in vivo and human clinical studies have demonstrated the association between celecoxib (Celebrex, pfizer, NY, USA), a specific cyclooxygenase 2 (COX-2) inhibitor and colorectal cancer (CRC) prevention. Nevertheless, the molecular mechanism responsible for the chemopreventive effect of celecoxib is not completely understood, and most probably involve several pathways. Multiple lines of evidence from in vitro studies and animal models indicate that celecoxib exert its effect by specifically inhibiting the COX-2 isoenzyme, that is up-regulated in 40-50% of colorectal polyps and in up to 85% of CRC. However, other studies have suggested that celecoxib may also inhibit polyps formation by COX-2 independent mechanisms (e.g. inhibition of NF-kB activation, or suppresion of PPARδ recepor complex to bind to DNA. Aim: To evaluate if down-regulation of PGE₂ production by celecoxib treatmemt is sufficient to inhibit cell growth or induce apoptosis in human colon carcinoma cell line (HT-29). Materials and Methods: HT-29 cells obtained from the American Type Culture Collection. Cells were exposed, for 72 hours, to different concentrations (0-50 μM) of celecoxib. Growth inhibition was assessed by Coulter counter. Cell viability was assessed by the ability of metabolically active cells to reduce tetrazolium salt (XTT) to coloured formazan compounds. Cell cycle and apoptosis were determined by two independent methods: FACS analysis and DAPI or propidium iodide staining. PGE₂ concentration was determined by a specific enzyme-linked immunoassay (R&D Biosuystem, USA). COX-1 and COX-2 expression was measured by western blotting analysis. All experiments were repeated at least three times and gave similar results. Results: Low concentration of celecoxib (5-10 μM) inhibited 95% of PGE₂ production. At the same time there was no significant change on cell viability, cell growth, or induction of apoptosis. At higher concentrations of celecoxib (>20 μM) a significant inhibition of cell growth and induction of apoptosis was seen, in a dose dependent maner. There were no changes in the levels of COX-1 and COX-2 proteins. Conclusions: Inhibition of PGE₂ production is an early, but not sufficient step in the mechanism of celecoxib mediated cell growth inhibition. It is suggested that other targets on the COX-2 enzyme, that are not correlated with PGE₂ production, or other independent COX-2 pathways are associated with the chemopreventive properties of celecoxib.