Volume 128, Issue 3, Pages 642-653 (March 2005) REG Iα protein may function as a trophic and/or anti-apoptotic factor in the development of gastric cancer  Akira Sekikawa, Hirokazu Fukui, Shigehiko Fujii, Jun Takeda, Apichart Nanakin, Hiroshi Hisatsune, Hiroshi Seno, Shin Takasawa, Hiroshi Okamoto, Takahiro Fujimori, Tsutomu Chiba  Gastroenterology  Volume 128, Issue 3, Pages 642-653 (March 2005) DOI: 10.1053/j.gastro.2004.12.045 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Immunostaining of (A) REG Iα protein and (B) PCNA in an early gastric cancer and its adjacent gastric mucosa (bars, 200 μm). REG Iα protein is expressed strongly not only in the cancerous lesion (CA) but also in the adjacent intestinal metaplasia (IM). Inflammatory cells have infiltrated the lamina propria. PCNA is expressed strongly in the nuclei of both cancerous and metaplastic cells. The distribution of PCNA-positive cells corresponds closely to that of REG Iα-positive cells. Immunohistochemical double staining for (C) REG Iα and PCNA, (D) Chromogranin A, (E) MUC1, and (F) MUC2 in the early gastric cancer tissues (bars, 10 μm). (C–F) Middle panels are merged photographs of REG Iα and the other markers. (C) Well-differentiated adenocarcinoma showing high co-expression of REG Iα and PCNA. Signals for REG Iα (green) are detectable in the cytoplasm, and the signals for PCNA (red) are detectable in the nuclei of cancer cells. (D) The invasive front of the gastric cancer tissue. A normal endocrine cell (top of the panels) is positive for both REG Iα and Chromogranin A, but the other cancer cells are positive only for REG Iα. (E) Signet-ring cell carcinoma showing partial co-expression (yellow) of REG Iα and MUC1. (F) Intestinal-type early gastric cancer showing high co-expression (yellow) of REG Iα and MUC2. Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 (A) Comparison of the PCNA labeling index between REG Iα-positive (n = 55) and -negative (n = 50) early gastric cancer tissues. The vertical lines represent the mean ± SEM. (B) Correlation between REG Iα-labeling index and PCNA-labeling index in early gastric cancer tissues (P < .01, r = 0.48). (C) Comparison of the severity of total inflammatory cell infiltration in the gastric mucosa adjacent to REG Iα-positive (n = 55) and -negative (n = 50) early gastric cancers. *Significantly greater than the REG Iα-negative group (P < .05). Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 REG Iα promoter activity in AGS and MKN74 cells in response to various cytokines. Cells were cotransfected with the hREG Iα(−1195/+78)-Luc construct and Renilla luciferase plasmid pRL-TK (as a control for transfection efficiency). Forty-eight hours later, the cells were stimulated with IFN-γ, TNF-α, IL-6, and IL-8. Luciferase activity was measured in extracts from transfected AGS and MKN74 cells and normalized for Renilla luciferase activity. All results are expressed relative to the activity of the untreated cell group at the 0-hour time point. The vertical lines represent the means ± SEM of 4 independent experiments. *P < .05 vs. control at the same time point. Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Effects of cytokines on REG Iα mRNA expression in AGS and MKN74 cells. Total RNA (20 μg) was extracted 12 hours after stimulation with cytokines and analyzed by Northern blotting using 32P-labeled cDNAs for REG Iα and glyceraldehyde-3-phosphate dehydrogenase mRNA. Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 (A) Effects of REG Iα gene induction on gastric cancer cell growth. AGS cells transfected with pIRES2-hREG Iα (AGS-REG Iα) or pIRES2-EGFP (AGS-EGFP; control) plasmid were cultured for 24, 48, and 72 hours. The WST-8 cleavage in each group was measured by enzyme-linked immunosorbent assay (absorbance OD450) as described in the Materials and Methods section. ●, AGS-REG Iα; ○, AGS-EGFP. (B) Effects of anti-REG Iα antibody on the cell growth of AGS-REG Iα and AGS-EGFP. AGS-REG Iα and AGS-EGFP cells were incubated with or without an anti-REG Iα antibody (Ab; 50 μg/mL) for 72 hours. (C) The source of the REG Iα protein. Human embryonic kidney 293T cells were transfected with a human REG Iα cDNA expression plasmid or a control plasmid, and the medium conditioned by these cells was collected. The release of REG Iα protein into the conditioned medium of the human REG Iα cDNA-transfected cells was confirmed by Western blot analysis with an anti-human REG Iα monoclonal antibody. (D) Effects of REG Iα-conditioned medium on the 5-bromo-2′-deoxyuridine incorporation of human gastric cancer cells. (E) Time-course effects of REG Iα-conditioned medium on gastric cancer cell growth. AGS cells were incubated with REG Iα or control medium for 24–72 hours. ●, REG Iα; ○, control medium. (F) Effects of anti-REG Iα antibody on the gastric cancer cell growth promoted by REG Iα protein. Anti-REG Iα antibody (Ab; 50 μg/mL) was added to control or REG Iα medium, followed by 72 hours of incubation. All results in A, B, and D–F are expressed as the means ± SEM of 4 samples. *Significantly greater than the control (AGS-EGFP or control medium-treated) group at the same time point (P < .01). #Significantly lower than the AGS-REG Iα or REG Iα-medium-treated group (P < .01). Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 (A) Resistance to H2O2-induced apoptosis of human gastric cancer cells transfected with REG Iα cDNA. AGS cells transfected with pIRES2-hREG Iα (AGS-REG Iα) or pIRES2-EGFP (AGS-EGFP; control) plasmid were treated with H2O2 (0.25–1.0 mmol/L) for 2 hours, followed by incubation in fresh culture medium for 24 hours. The percentage of TUNEL-positive cells was evaluated as described in the Materials and Methods section. □, AGS-EFGP; ■, AGS-REG Iα. (B) Effects of anti-REG Iα antibody (Ab; 50 μg/mL) on H2O2 (0.5 mmol/L)-induced apoptosis of AGS-REG Iα and AGS-EGFP cells. (C) Resistance to H2O2-induced apoptosis in gastric cancer cells treated with REG Iα-conditioned medium. AGS cells were incubated with the conditioned medium containing human-recombinant REG Iα or control medium for 24 hours, followed by a 2-hour incubation with the medium containing H2O2 (0.25–1.0 mmol/L). □, Control medium; ■, REG Iα medium. (D) Effects of anti-REG Iα antibody on H2O2 (0.5 mmol/L)-induced apoptosis of AGS cells. Anti-REG Iα antibody (Ab; 50 μg/mL) was added to control or REG Iα-conditioned medium. All results are expressed as the means ± SEM of 4 samples. *P < .05, **P < .01 vs. control (AGS-EGFP or control medium-treated) group at the same dose point. #Significantly higher than the AGS-REG Iα or REG Iα medium-treated group (P < .01). Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Effects of REG Iα-conditioned medium on caspase activity of human gastric cancer cells treated with (A) H2O2 or (B) serum withdrawal. All results are expressed as the means ± SEM of 4 samples. *P < .05 vs. control group at (A) the same dose or (B) time point. □, Control medium; ■, REG Iα medium. Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Effects of REG Iα-conditioned medium on the phosphorylation of Akt and the expression of Bcl family proteins in gastric cancer cells. Western blotting indicated that the phosphorylation of (A) Akt and the expression of (B) Bcl-xL but not Bcl-2 or Mcl-1 were enhanced in AGS cells after 12 hours of incubation with REG Iα-conditioned medium. Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 (A) Effects of REG Iα gene induction on gastric cancer cell survival. AGS cells transfected with pIRES2-hREG Iα (AGS-REG Iα) or pIRES2-EGFP (AGS-EGFP; control) plasmid were treated with H2O2 (0.25–1.0 mmol/L) for 2 hours, followed by incubation in fresh culture medium for 24 hours. The percentage of viable cells was evaluated as described in the Materials and Methods section. ●, AGS-REG Iα; ○, AGS-EGFP. (B) Effects of anti-REG Iα antibody (Ab; 50 μg/mL) on H2O2 (0.5 mmol/L)-induced cell death in AGS-REG Iα and AGS-EGFP cells. (C) Effects of REG Iα-conditioned medium on gastric cancer cell survival. AGS cells preincubated with REG Iα or control medium were treated similarly with H2O2 (0.25–1.0 mmol/L) for 2 hours. ●, REG Iα medium; ○, control medium. (D) Effects of anti-REG Iα antibody (Ab; 50 μg/mL) on H2O2 (0.5 mmol/L)-induced cell death of AGS cells. All results are expressed as the means ± SEM of 4 samples. *P < .05, **P < .01 vs. control (AGS-EGFP or control medium-treated) group at the same dose point. #Significantly lower than the AGS-REG Iα or REG Iα medium-treated group (P < .05). Gastroenterology 2005 128, 642-653DOI: (10.1053/j.gastro.2004.12.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions