Volume 136, Issue 3, Pages 953-966 (March 2009) Zinc Replenishment Reverses Overexpression of the Proinflammatory Mediator S100A8 and Esophageal Preneoplasia in the Rat  Cristian Taccioli, Shao–Gui Wan, Chang–Gong Liu, Hansjuerg Alder, Stefano Volinia, John L. Farber, Carlo M. Croce, Louise Y.Y. Fong  Gastroenterology  Volume 136, Issue 3, Pages 953-966 (March 2009) DOI: 10.1053/j.gastro.2008.11.039 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Expression profiling of zinc-modulated rat esophageal mucosa. (A) Dendrogram illustrating clustering of 30,000 transcripts in ZD, ZR, and ZS esophagi. (B) A total of 103 genes in ZD vs ZS esophagi (fold-change ≥ 4). (C) Scatterplot of ZD vs ZS esophagi. (A–C) Red and green denote up-regulated and down-regulated genes; n = 4/group. (D) H&E-stained sections of normal ZS, hyperplastic ZD, and restored ZR esophagus; scale bar = 25 μm. Gastroenterology 2009 136, 953-966DOI: (10.1053/j.gastro.2008.11.039) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Validation of array data. (A) Array data showing that ZR restores the altered expressions of 14 genes in ZD esophagi to ZD/ZS approximately 1, and S100A8 to ZD/ZS less than 2 levels. (B) qRT-PCR validation for 7 genes. (C) Immunoblots (n = 3/group) for 6 genes and glyceraldehyde-3-phosphate dehydrogenase (gapdh) (normalizer). P < .001 (S100A8, ZD vs ZS; ZD vs ZR); P < .01 (SERPINB3, PTGES, and CSRP3, ZD vs ZS; ZD vs ZR); and P < .05 (DNMT3A and PPP2R1A, ZD vs ZS). (D) Fold-change of protein expression above ZS. Values are mean ± SEM. Gastroenterology 2009 136, 953-966DOI: (10.1053/j.gastro.2008.11.039) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Spatial and temporal localization of S100A8 and RAGE protein (red, 3-amino-9-ethylcarbazole substrate-chromogen) in near-serial sections of archived esophageal tissues from zinc-modulated rats that overexpressed NF-κΒ p65 and COX-2 (brown, 3,3′-diaminobenzidine tetrahydrochloride). (A, E, and I) S100A8 and (B, F, and J) RAGE were intensely and abundantly co-overexpressed in ZD esophagus, and occurred in similar spatial patterns as those of (C, G, and K) NF-κΒ p65 and (D, H, and L) COX-2. The four proteins show similar spatial patterns (A–D, and arrows in I–L) (M) 100A8 and (N) RAGE expression was greatly reduced at 48 hours after ZR, as was (O) NF-κΒ p65 and (P) COX-2. ZS esophagi showed sporadic and weak expression (arrows) of (Q) S100A8 and (R) RAGE, and weak or absent staining of (S) NF-κΒ p65 and (T) COX-2. Scale bars, (A–D and Q–T) 100 μm; (I–P) 50 μm; (E–H, bracketed area in A–D) 25 μm. C, G, and K were reproduced from the Int J Cancer, Vol 122, No 5, 2008, pgs 978–989. ©2008 John Wiley & Sons, Inc. Reprinted with permission from John Wiley & Sons, Inc. D, H, and L were reproduced from Fong et al7 with permission. Gastroenterology 2009 136, 953-966DOI: (10.1053/j.gastro.2008.11.039) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 (A) Effects of celecoxib and zinc on S100A8 and RAGE protein expression. S100A8 and RAGE protein (red, 3-amino-9-ethylcarbazole substrate-chromogen) immunostaining was (A1, A2) intense in hyperplastic ZD esophagi at 8 hours after celecoxib treatment, but (A3, A4) sporadic/weak after zinc treatment. Scale bars, 50 μm. (B) Correlation of S100A8/A9 mRNA expression levels (normalized to glyceraldehyde-3-phosphate dehydrogenase; values are mean ± SEM; n = 6–8/group) with esophageal tumorigenic outcome in NMBA-treated zinc-modulated rats. S100A8 (ZD vs ZS: 0 hour, 5 and 15 wk; ZD vs ZR: 5 and 15 wk; P < .001); S100A9 (ZD vs ZS: 0 hour, 5 and 15 wk; ZD vs ZR: 5 and 15 wk; P < .01). Inset: tumor incidence: ZD (100%) vs ZS (16.6%), P = 7.8 × 10−6; ZD (100%) vs ZR (28.9%); P = 5.0 × 10−5. Multiplicity: ZD (11 ± 3.8) vs ZS (0.5 ± 0.3) and ZD (11 ± 3.8) vs ZR (0.6 ± 0.4); P < .001. Gastroenterology 2009 136, 953-966DOI: (10.1053/j.gastro.2008.11.039) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 S100A8 protein expression in (A–D) human and (E–F) mouse ESCC. (A–C) Moderate to intense S100A8 immunostaining (red, 3-amino-9-ethylcarbazole substrate-chromogen) in tumor tissues and stroma (arrows), and (D) very weak staining in adjacent near-normal esophageal mucosa in an ESCC section. (E and F) ESCC from 4-nitroquinoline 1-oxide–treated zinc-deficient p53+/− mice. (E) Intense and (F) moderately strong cytoplasmic S100A8 staining occurred in invasive tumor areas and stroma (arrows). Scale bars: (A–D, F) 25 μm and (E) 50 μm. Gastroenterology 2009 136, 953-966DOI: (10.1053/j.gastro.2008.11.039) Copyright © 2009 AGA Institute Terms and Conditions