Influence of the nitric oxide donor glyceryl trinitrate on apoptotic pathways in human colon cancer cells

Background & Aims: We have previously reported the role of nitric oxide in colon tumor regression in vivo. The present study was designed to explore the influence of an endogenous nitric oxide donor, glyceryl trinitrate (GTN), on cell death pathways in colon cancer cells. Methods: Human colon cancer cell lines were treated with the NO donor GTN. Apoptosis was identified by morphological criteria and the terminal deoxynucleotidyl transferase-mediated deoxyuridine (TUNEL) method. The mitochondrial transmembrane potential was studied by flow cytometry, cytochrome c release by Western blot, and caspase activation by combining fluorogenic peptide substrates, peptide inhibitors, and immunoblotting. Expression of death receptors was studied by flow cytometry and confocal microscopy. Results: GTN induces a dose- and time-dependent cell death by apoptosis in colon cancer cells. This cell death pathway involves the mitochondria and caspases, mainly caspase-1 and caspase-10. In contrast, caspase-3 activation is a late and limited event. Death receptors are not involved in GTN-mediated cell death, while GTN sensitizes tumor cells to Fas-ligand-induced apoptosis. This permissive effect correlates with an increased expression of Fas receptor and a decreased expression of several endogenous inhibitors of apoptosis (IAPs). Conclusions: Our results indicate that GTN (1) activates an unusual caspase cascade to induce apoptosis in colon cancer cells and (2) sensitizes these cells to Fas-mediated cell death by increasing the expression of Fas and decreasing the expression of several IAPs.