Volume 126, Issue 1, Pages 136-147 (January 2004) Cyclooxygenase-2 inhibitor (SC-236) suppresses activator protein-1 through c-Jun NH2- terminal kinase  Benjamin Chun-yu Wong, Xiao Hua Jiang, Marie C.M. Lin, Shui Ping Tu, Jian Tao Cui, Shi Hu Jiang, Wai Man Wong, Man Fung Yuen, Shiu Kum Lam, Hsiang Fu Kung  Gastroenterology  Volume 126, Issue 1, Pages 136-147 (January 2004) DOI: 10.1053/j.gastro.2003.10.063

Figure 1 Effects of SC-236 on anchorage-independent cell growth and PMA-induced cell transformation. (A) 104 Gastric cancer cells were exposed to different concentrations of SC-236 in 0.33% agar for 14 days and scored for colonies at the end of the experiment. (B) 104 JB6 cells were exposed to different concentrations of SC-236 or aspirin in the presence or absence of 100 nmol/L PMA in 0.33% agar for 14 days and scored for colonies at the end of the experiment. (C) 5 × 103 JB6 cells were plated on 96-well plates and treated with different concentrations of SC-236 for 24 hours. MTT assay was performed. All results were expressed as the mean of 3 independent experiments ± standard error. Gastroenterology 2004 126, 136-147DOI: (10.1053/j.gastro.2003.10.063)

Figure 2 SC-236 suppressed AP-1 transcriptional and binding activity. (A) Cells transiently expressing AP-1 luciferase reporter gene construct were treated with different concentrations of SC-236 for 2 hours then followed with 100 nmol/L PMA for another 24 hours. Cells were then harvested for analysis of luciferase activity. The firefly luciferase reading was normalized to renilla luciferase reading. Results were expressed as the means of 3 independent experiments ± standard error. (B) AGS cells were treated with 50 μmol/L SC-236 alone for 2 hours or pretreated with SC-236 for 1 hour, followed by 100 nmol/L PMA for another 1 hour. Nuclear extracts were prepared and analyzed in an electrophoretic mobility shift assay with a DIG-labeled AP-1 probe. Equal amounts (6 μg) of nuclear protein were loaded in each lane. In lane 5, a 50-fold excess of unlabeled oligonucleotide was added before the addition of DIG-labeled probe. Gastroenterology 2004 126, 136-147DOI: (10.1053/j.gastro.2003.10.063)

Figure 3 SC-236 inhibited PMA-induced JNK phosphorylation and activity. AGS cells were pretreated with different concentrations of SC-236 for 2 hours then exposed to PMA for another 1 hour. Total and phosphorylated proteins were detected with Western blot assay. (A) SC-236 inhibited c-Jun phosphorylation. (B) SC-236 inhibited JNK phosphorylation. Quantification of phosphorylation of c-Jun, ERK, and JNK was performed by scanning densitometry of the bands, and the values shown are the means ± SE (n = 3) and are expressed as percentages of the maximal increase above the control values. (C) The effect SC-236 and aspirin on JNK kinase activity. JNK activity was determined by an in vitro kinase assay. The JNK activity kit utilized a JNK-specific antibody to immunoprecipitate JNK from cell lysates. Activity of the JNK was then determined in a kinase reaction using recombinant c-Jun as substrate. Phosphorylation of the c-Jun was analyzed by Western blot analysis using a phospho-c-Jun specific antibody. The right panel showed the values (mean ± SE, n = 3) of the level of JNK activation by in vitro kinase assay obtained from scanning densitometry expressed as a percentage of the maximum increase. ∗P < 0.05. Gastroenterology 2004 126, 136-147DOI: (10.1053/j.gastro.2003.10.063)

Figure 4 SC-236 inhibited JNK-c-Jun pathway. (A) AGS cells were transiently cotransfected with pFR-Luc and pFA2-c-Jun plasmid in the presence or absence of pFC-MEKK and treated with 25 μmol/L SC-236 for 2 hours followed with 100 nmol/L PMA for another 12 hours. Cells were then harvested for analysis of luciferase activity. Results were expressed as the means of 3 independent experiments ± standard error. (B). Top panel: Western blot analysis of phospho-JNK. Cells were pretreated with or without 2.5 μmol/L SP600125 for 1 hour, followed with PMA for another 1 hour. Bottom panel: AP-1 transcriptional activity assay. Cells were pretreated with or without 2.5 μmol/L SP600125 for 1 hour and then treated with SC-236 for 2 hours, followed with PMA for another 24 hours. At the end of the experiments, cells were harvested for analysis of luciferase activity. (C) Top panel: Western blot analysis of phospho-ERK. Cells were pretreated with or without 10 μmol/L U0126 for 1 hour, followed with PMA for another 1 hour. Bottom panel: AP-1 transcriptional activity assay. Cells were pretreated with or without 10 μmol/L U0126 for 1 hour and then treated with SC-236 for 2 hours, followed with PMA for another 24 hours. At the end of the experiments, cells were harvested for analysis of luciferase activity. ∗P < 0.01, #P > 0.05, compared with PMA treatment without SC-236, separately. Gastroenterology 2004 126, 136-147DOI: (10.1053/j.gastro.2003.10.063)

Figure 5 Suppression of JNK activity inhibited gastric cancer cell growth. (A) Total protein was extracted from cells 48 hours following treatment with 0.4 μmol/L JNK1AS, 0.4 μmol/L JNK2AS, 0.2 μmol/L JNK1 AS + 0.2 μmol/L JNK2AS, or control oligonucleotides (JNKScr). Protein samples were analyzed by Western blot analysis using specific JNK antibody. (B) Total Jun kinase activity was determined 1 hour following exposure to PMA by an in vitro kinase assay. Forty-eight hours before PMA treatment, cells were transfected with JNK1 AS, JNK2AS, combinations of JNK1AS plus JNK2AS (0.4 μmol/L each), or JNKScr. (C) JNKAS inhibited cell growth in AGS cells. Proliferation assays were performed as described in the Materials and Methods section. The cells were maintained in 2% FBS during the experiment. Five days after the treatment, the cells were counted with a Coulter counter. The proliferation data shown here were the average of 2 identical and independent experiments, each carried out in triplicate. ∗P < 0.05. Gastroenterology 2004 126, 136-147DOI: (10.1053/j.gastro.2003.10.063)

Figure 6 The effect of SC-236 on COX-2 activity and PGE2 production. (A) Cells were plated on 24-well plates and exposed to PMA with or without SC-236 for 24 hours. COX-2 activity was determined as described in the Materials and Methods section. Results are mean ± SD of triplicate assays and 2 experiments. (B) Cells were plated on 24-well plates and exposed to PMA with or without SC-236 for 24 hours. PGE2 content of the conditioned media was determined by ELISA. Results are mean ± SD of triplicate assays and 2 experiments. ∗P < 0.05. Gastroenterology 2004 126, 136-147DOI: (10.1053/j.gastro.2003.10.063)

Figure 7 Exogenous prostaglandins PGE2, PGH1, and PGH2 had no effect on cell growth and AP-1 activation. (A) AGS cells were plated on 24-well plates and exposed to 100 μmol/L NS-398 or 100 μmol/L Nimesulide or 100 μmol/L sunlindac sulfone for 2 hours, followed with PMA for another 12 hours. The AP-1 luciferase enzyme activity was measured using the luminometer. (B) AGS cells were pretreated with 100 μmol/L sulindac sulfone or 25 μmol/L SC-236 or 100 μmol/L NS-398 followed with PMA for another 1 hour. Total and phosphorylated proteins were detected with Western blot assay. (C) 104 AGS cells were exposed to 2 μg/mL of different prostaglandins in 0.33% agar for 14 days and scored for colonies at the end of the experiment. (D) AGS cells were plated on 24-well plates and exposed to different concentrations of prostaglandins with SC-236 for 2 hours, followed with 100 nmol/L PMA for another 12 hours. The AP-1 luciferase enzyme activity was measured using the luminometer. ∗P > 0.05. Gastroenterology 2004 126, 136-147DOI: (10.1053/j.gastro.2003.10.063)