Volume 123, Issue 1, Pages 235-246 (July 2002) Influence of the nitric oxide donor glyceryl trinitrate on apoptotic pathways in human colon cancer cells  Anne Millet, Ali Bettaieb, Flore Renaud, Laurent Prevotat, Arlette Hammann, Eric Solary, Bernard Mignotte, Jean–Francois Jeannin  Gastroenterology  Volume 123, Issue 1, Pages 235-246 (July 2002) DOI: 10.1053/gast.2002.34310 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 GTN triggers apoptosis in human colon tumor cell lines. (A) Exponentially growing SW480, SW620, and HCT116 cells (2 × 105/mL) were treated with indicated concentrations of GTN for 48 hours at 37°C. Apoptotic cells were identified by fluorescence microscopy after nuclei staining with Hoechst 33342. (B) Morphological aspect of SW480 cells ongoing apoptosis as observed by fluorescence microscopy. (S), Floater cells collected in the supernatant. (C and D) SW480 cells were treated with indicated doses or for indicated times with GTN before counting apoptotic cells as above. Results are the mean ± SD of 3 independent experiments. (E) Western blot analysis of PARP in SW480 cells exposed to 500 μmol/L GTN for 48 hours (1 representative of 3 independent experiments). Gastroenterology 2002 123, 235-246DOI: (10.1053/gast.2002.34310) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Mitochondrial changes during GTN-induced colon cancer cell apoptosis. (A) SW480 cells were treated with 500 μmol/L GTN for indicated times, then stained with 1 nmol/L DiOC6(3) probe before measuring cell fluorescence by flow cytometry (FL-1). Diminished mitochondrial membrane bound DiOC6(3) (reduced FL-1 intensity) indicates a decrease in Δψm. (B) Untreated (control) and GTN-treated (500 μmol/L for 24 hours) cells were separated into mitochondrial and cytosolic fractions to determine cytochrome c subcellular localization by Western blot analysis. Gastroenterology 2002 123, 235-246DOI: (10.1053/gast.2002.34310) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Involvement of caspases in GTN-induced colon cancer cell apoptosis. SW480 and HCT116 cells (2 × 105/mL) were pretreated for 1 hour with 100 μmol/L caspase inhibitors before exposure to 500 μmol/L GTN for 48 hours. Apoptotic cells were counted after Hoechst 33342 staining. Results are the mean ± SD of 3 independent experiments. *P < 0.05. Gastroenterology 2002 123, 235-246DOI: (10.1053/gast.2002.34310) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of GTN treatment on caspase activation. (A) SW480 and HCT116 cells were treated with 500 μmol/L GTN for indicated times before measuring caspase activities. Mean ± SD of 3 independent experiments. (B and D) Western blot analysis of indicated proteins in SW480 cells exposed for indicated times (h) to 500 μmol/L GTN. Actin: loading control. (S), Floating cells collected in the supernatant. (C and D) SW480 cells were treated with 500 μmol/L GTN in the absence or presence of 100 μmol/L caspase-10 inhibitor (Z-AEVD-fmk) for 24 hours before measuring (C) caspase-3 and -10 activities (one representative of 3 independent experiments in triplicate) and (D) Western blot analysis of caspase-3. *P < 0.05. Gastroenterology 2002 123, 235-246DOI: (10.1053/gast.2002.34310) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of GTN on caspase-3 activation by cytochrome c/dATP. Equal amounts of cell-free extracts from untreated and treated (500 μmol/L GTN for 24 hours) SW480 cells were incubated with 5 mmol/L cytochrome c and 1 mmol/L dATP. Caspase-3 activity was measured using the fluorogenic caspase substrate, Ac-DEVD-AMC. One representative of 3 independent experiments in triplicate is shown. Gastroenterology 2002 123, 235-246DOI: (10.1053/gast.2002.34310) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) GTN sensitizes colon cancer cells to Fas-mediated apoptosis. SW480 and HCT116 cells were treated with 500 μmol/L GTN for 24 hours at 37°C, followed by a 24-hour incubation with the soluble recombinant-Fas ligand (0.5 AU/mL). When indicated, SW480 cells were preincubated with 100 μmol/L caspase inhibitor for 30 minutes or with 500 ng/mL ZB4 anti-Fas antagonistic antibody for 1 hour. (A and B) Apoptotic cells were counted after Hoechst staining. Mean ± SD of 3 independent experiments. (C) Western blot analysis of PARP cleavage realized on SW480 cells (1 representative of 3 experiments). *P < 0.05. Gastroenterology 2002 123, 235-246DOI: (10.1053/gast.2002.34310) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of GTN on Fas receptor expression and clustering. (A) Flow cytometry analysis of Fas expression at the surface of SW480 cells left untreated (hatched line) or treated with 500 μmol/L GTN for 48 hours at 37°C (bold line). A nonimmune mouse IgG1 was used as control (green curve). (B) Confocal laser scanning microscopy analysis of Fas expression in SW480 and HCT116 cells treated with 500 μmol/L GTN for indicated times. One representative of 3 experiments is shown. Gastroenterology 2002 123, 235-246DOI: (10.1053/gast.2002.34310) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of GTN on the expression of FLIP and the IAP protein levels. Western blot analysis of c-FLIP and indicated IAPs in SW480 cells treated with 500 μmol/L GTN for indicated times. Actin: loading control. One representative of 3 experiments. Gastroenterology 2002 123, 235-246DOI: (10.1053/gast.2002.34310) Copyright © 2002 American Gastroenterological Association Terms and Conditions