Volume 138, Issue 1, Pages 221-230 (January 2010) Inhibition of Colon Carcinogenesis by 2-Methoxy-5-Amino-N-Hydroxybenzamide, a Novel Derivative of Mesalamine  Carmine Stolfi, Massimiliano Sarra, Roberta Caruso, Massimo Claudio Fantini, Daniele Fina, Roberto Pellegrini, Giampiero Palmieri, Thomas T. Macdonald, Francesco Pallone, Giovanni Monteleone  Gastroenterology  Volume 138, Issue 1, Pages 221-230 (January 2010) DOI: 10.1053/j.gastro.2009.08.062 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 The 2-14–mediated inhibition of CRC cell growth is associated with an accumulation of cells in G0/G1 phase of cell cycle. (A) 2-14 inhibits the growth of HCT-116 and HT-29 cells. Carboxyfluorescein diacetate succinimidyl ester–labeled cells were either left untreated (Untr) or treated with 2-14 for 24 hours and then evaluated by flow cytometry. Data indicate mean ± SD of 4 experiments (HCT-116: untreated vs 2-14–treated cells, *P = .02, **P = .005, ***P < .001; HT-29: untreated vs 2-14–treated cells, †P = .006, ††P < .001). Right insets. Representative histograms of HCT-116 cells left untreated or treated with 2-14. Numbers above lines indicate the percentages of proliferating cells. (B) 2-14–treated cells accumulate in G0/G1 phase. HCT-116 cells were treated or not with 2-14 for 24 hours. Values are the percentages of cells in the different phases of cell cycle and indicate mean ± SD of 4 experiments (untreated vs 2-14–treated cells, *P < .04, **P < .001). Right insets show representative dot-plots of the cell-cycle distribution. (C) 2-14 down-regulates cyclin D1 protein expression. Cells were either left untreated or treated with 2-14, and protein extracts were analyzed by Western blotting. (D) 2-14 does not reduce cyclin D1 RNA expression. Cells were treated or not with 2-14 for the indicated time points; cyclin D1 RNA was evaluated by real-time polymerase chain reaction. Levels are normalized to β-actin. Values are mean ± SD of 3 experiments. (E) 2-14 enhances CDK2 phosphorylation at Thr-14 and Tyr-15 residues. HCT-116 cells were treated with or without 2-14, and p-CDK2 and total CDK2 were analyzed by Western blotting. One of 4 representative blots is shown. Gastroenterology 2010 138, 221-230DOI: (10.1053/j.gastro.2009.08.062) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 2-14 induces ERS. (A) Representative Western blots for p-eIF2α Ser51 and total eIF2α in extracts of HCT-116 cells either left untreated (Untr) or treated with 2-14. One of 4 representative experiments is shown. (B) 2-14 enhances ATF4 RNA expression. HCT-116 cells were treated or not with 2-14 for 8 hours, and ATF4 transcripts were evaluated by real-time polymerase chain reaction. Thapsigargin (tg) was used as a positive control for ERS induction. Levels are normalized to β-actin. Values are mean ± SD of 4 experiments (untreated cells vs 2-14–treated cells, *P = .02, **P < .001; untreated cells vs TG-treated cells, **P < .001). (C) 2-14 promotes splicing of XBP-1 mRNA. HCT-116 cells were either left untreated or treated with 2-14 for 8 hours. XBP-1 splicing was evaluated by reverse transcription polymerase chain reaction. One of 3 separate experiments is shown. us, unspliced form of XBP-1; s, spliced form of XBP-1; gapdh, glyceraldehyde-3-phosphate dehydrogenase. Gastroenterology 2010 138, 221-230DOI: (10.1053/j.gastro.2009.08.062) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Involvement of PERK in the 2-14–mediated cell-cycle arrest. (A) 2-14 enhances the phosphorylation of PERK on Thr980 residue. Representative Western blots for p-PERK Thr980 and total PERK in extracts of HCT-116 cells either left untreated (Untr) or treated with 2-14. One of 3 representative experiments is shown. (B) Silencing of PERK significantly reduces the 2-14–induced cyclin D1 protein decline. Representative Western blots for PERK, cyclin D1, and β-actin in extracts of HCT-116 cells transfected with PERK or control siRNA for 2 days and then either left untreated or treated with 2-14 for 8 hours. Right insets: representative dot-plots showing the fractions of transfected cells. HCT-116 cells were transfected with fluorescein-conjugated or nonconjugated control siRNA for 2 days, and the percentage of fluorescein isothiocyanate (fitc)-labeled cells was assessed by flow cytometry. (C) Percentages of PERK or control siRNA-transfected HCT-116 cells accumulated in G0/G1 phase after treatment with 2-14. Cells were transfected with PERK or control siRNA as described earlier and, after 24 hours, either left untreated or treated with 2-14 for a further 24 hours. Data indicate mean ± SD of 3 separate experiments (*P < .01). Gastroenterology 2010 138, 221-230DOI: (10.1053/j.gastro.2009.08.062) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 2-14 induces CRC cell death. (A) Representative histograms showing the percentages of Annexin V (av) and/or PI-positive HCT-116 cells after treatment with 2-14 for 24 (upper panel) and 48 hours (lower panel). Data are mean ± SD of 5 experiments. (B) 2-14 activates caspase-3 in HCT-116. Cells were treated with 2-14, and activation of caspase-3 was assessed by flow cytometry. (C and D) Q-VD-OPH, a pan-caspase inhibitor, blocks caspase-3 activation and cell death induced by 2-14. Cells were cultured in the presence or absence of Q-VD-OPH for 1 hour, and then either left untreated or treated with 2-14 for 36 and 48 hours, respectively. The percentages of caspase-3-, AV-, and PI-positive cells were assessed by flow cytometry. Results are mean ± SD of 4 experiments. (E) The 2-14–induced cell growth arrest is not secondary to induction of cell death. HCT-116 cells were cultured in the presence or absence of Q-VD-OPH for 1 hour, and then treated or not with 2-14 for 24 hours. The percentage of proliferating cells was assessed by flow cytometry. Results are mean ± SD of 3 experiments. Gastroenterology 2010 138, 221-230DOI: (10.1053/j.gastro.2009.08.062) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 (A) 2-14 reduces the in vivo formation of CT26-derived tumors. CT26 were inoculated into Balb/c mice, and animals were treated daily with subcutaneous injection of 2-14 (1–12 mg/kg/mouse) starting 3 days after the CT26 injection. Representative photographic images of xenografts developed in control (Ctr) and in 2-14–treated mice are shown at the bottom of the respective histograms (control vs 2-14–treated mice, *P < .01, **P < .001). (B) 2-14 inhibits CT26-derived xenografts when administered intraperitoneally. Control mice received daily PBS. Data indicate mean ± SD of all experiments (control vs 2-14–treated mice, *P < .001). (C) 2-14 reduces cyclin D1 protein expression in the CT26-derived tumors. Representative Western blots showing cyclin D1 in extracts of xenografts treated with PBS (Ctr) or with 2 different doses of 2-14. One of 3 separate experiments in which similar results were obtained is shown. (D) Subcutaneous administration of 2-14 reduces the size of stabilized CT26-derived tumors. CT26-derived tumors were induced as described earlier. At day 14, mice were allocated to receive daily subcutaneous PBS, mesalamine, or 2-14 for a further 2 weeks. Data indicate mean ± SD of all experiments (control vs 2-14–treated mice; *P = .003). Gastroenterology 2010 138, 221-230DOI: (10.1053/j.gastro.2009.08.062) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 2-14 reduces the development of azoxymethane/DSS colitis-driven neoplastic lesions. (A) Representative endoscopic pictures showing polypoid lesions in mice treated with PBS (Ctr) or 2-14. Right inset shows the endoscopic scoring of tumors in mice treated with PBS or 2-14. Data indicate the mean ± SD of all experiments in which at least 6 mice per group were considered (control vs 2-14–treated mice, *P = .03). (B) Representative pictures of H&E-stained colon sections of mice either treated with PBS (control) or 2-14 (original magnification, 40×). A polypoid or flat lesion is seen in control and 2-14–treated mice, respectively. Bottom left inset shows a representative H&E-stained colonic section containing a dysplastic area in a mouse treated with PBS (400×). Bottom right inset shows a neoplastic area infiltrating a colonic follicle in a mouse treated with PBS. (C) Effect of 2-14 treatment on the multiplicity of neoplastic lesions. Tumors were induced as described earlier and mice were treated with 2-14 or PBS. At the end of the experiment, polypoid and flat lesions were counted (*P = .001; **P = .02). Gastroenterology 2010 138, 221-230DOI: (10.1053/j.gastro.2009.08.062) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 2-14 reduces the proliferation of neoplastic but not normal epithelial cells. (A) Proliferating cell nuclear antigen immunostaining of colonic sections taken from mice with azoxymethane/DSS-induced neoplasia and treated with PBS or 2-14 (original magnification, 40× or 400× in the right lower inset in the left panel). (B) Representative photomicrographs (100×) of proliferating cell nuclear antigen–stained normal colonic sections taken from mice injected with azoxymethane/DSS and then treated with PBS or 2-14. Gastroenterology 2010 138, 221-230DOI: (10.1053/j.gastro.2009.08.062) Copyright © 2010 AGA Institute Terms and Conditions