Dietary iron enhanced cyclin D1 expression and promoted epithelial cell apoptosis during acute colonic inflammation.

<p>Colonic mRNA expression of cell cycle regulators, cyclin D1 (A), cyclin B1 (B) and c-myc (C) were measured in Iron and Control (Ctrl) mice treated with or without DSS for 7 days. PCNA protein expression (D) was determined by immunoblot in isolated colonic epithelial cells from Iron/DSS and Ctrl/DSS mice compared with non-DSS treated mice at day 3. Apoptotic colonic epithelial cells were quantified (E) from TUNEL staining (F; representative images shown) of whole colonic Swiss roll preparations from Iron and Ctrl mice treated with or without DSS for 3 days. Results are expressed as mean±SEM, n = 3–8. * <i>P</i><0.05 denotes significance between DSS treatment versus no treatment; # <i>P</i><0.05 denotes significance between Iron/DSS versus Ctrl/DSS or Iron versus Ctrl mice. @ <i>P</i><0.05 denotes significance between Iron/DSS versus Ctrl. Scale bar denotes 50 µm.</p

Cytokine gene expression in Iron and Control mice treated with or without DSS.

<p>Results are normalized against Hprt or Arbp expression and expressed relative to Control mice as mean±SEM; n = 3–6. * <i>P</i><0.05; denotes significance between DSS treatment versus no treatment and # <i>P</i><0.05; denotes significance between Iron/DSS versus Control/DSS or Iron versus Control mice.</p

Iron accumulation in the colon increased F₂-isoprostane levels.

<p>3,3′-diaminobenzidine-enhanced Perls' Prussian blue (A) and ferritin and F4/80 double immunofluorescent (B) staining were performed on colon sections from Iron and Control (Ctrl) mice treated with or without DSS for 3 days. Colonic iron (A) accumulated in apical epithelial cells (black arrowheads) and macrophages of the lamina propria (black arrows). Immunofluorescent staining (B) of ferritin (red) was increased in colonic epithelial cells (white arrowheads) and in lamina propria F4/80⁺ (green) macrophages (white arrows) in iron-supplemented mice. Colon F₂-isoprostane concentration (C), plasma transferrin saturation (D), liver non-heme iron concentration (E) were measured in Iron and Ctrl mice treated with or without DSS for 7 days. Results are expressed as mean±SEM, n = 4–7. * <i>P</i><0.05 denotes significance between DSS treatment versus no treatment; # <i>P</i><0.05 denotes significance between Iron/DSS versus Ctrl/DSS or Iron versus Ctrl mice. Scale bar denotes 25 µm.</p

Dietary iron enhanced acute colonic inflammation.

<p>Body weight (A), endoscopic MEICS (B) and histological colitis (C) scores were measured in Iron and Control (Ctrl) mice treated with or without DSS for up to 7 days. In hematoxylin and eosin-stained distal colon sections (D), Iron/DSS mice exhibited extensive areas of mucosal erosion (arrowheads) with severe crypt damage (arrows) whilst crypt damage was moderate in Ctrl/DSS mice. There was no damage in Iron and Ctrl mice. Results are expressed as mean±SEM, n = 6–8. * <i>P</i><0.01 denotes significance between DSS treatment versus no treatment; # <i>P</i><0.05 denotes significance between Iron/DSS versus Ctrl/DSS or Iron versus Ctrl mice. Scale bar denotes 50 µm.</p

Iron transporter gene expression was modified in colonic tumors in inflammation-associated tumorigenesis.

<p>Iron importer, Dmt1 (A) and Tfr1 (B), as well as iron exporter, Fpn (C), mRNA expression were measured in colonic tumor (T) and non-tumor (nT) tissue from Iron/DSS and Control (Ctrl)/DSS mice at day 78. 3,3′-diaminobenzidine-enhanced Perls' Prussian blue staining (D) showed that no iron deposits were detected in the tumor epithelium from Iron/DSS or Ctrl/DSS mice. In surrounding non-tumor tissue, there was some stainable iron detected in colonic epithelial cells (arrowheads) of Iron/DSS mice compared with Ctrl/DSS mice and there was some evidence of distension and branching of glands in the mucosa (D; arrows). Results are expressed as mean±SEM, n = 3–6. * <i>P</i><0.05 denotes significance between T and nT tissue from Iron/DSS or Ctrl/DSS mice; # <i>P</i><0.05 denotes significance between Iron/DSS and Ctrl/DSS in T or nT tissue. Scale bar denotes 25 µm.</p

Mouse primers.

<p>Mouse primers.</p

Dietary iron enhanced colonic inflammatory cytokine release and Stat3 signaling during acute colonic inflammation.

<p>Colonic IL-6 (A), IL-11 (B) and IL-1β (C) release from colonic tissue explants were measured after 24 hours in culture at 37°C. The tissue explants were harvested Iron and Control (Ctrl) mice treated with or without DSS for 7 days. Colonic Stat3 phosphorylation (D) was also determined in Iron/DSS and Ctrl/DSS mice compared with non-DSS treated mice at day 7. Immunofluorescent staining (E) of F4/80 (green) and IL-6 (red; left panels) or phosphorylated Stat3 (red; right panels) at day 3 of DSS treatment identified F4/80⁺ macrophages (arrows) as a main source of IL-6 production and epithelial crypt cells as active sites of nuclear pStat3 localization (white arrowheads) in Iron and Ctrl mice. Results are expressed as mean±SEM, n = 3–8. * <i>P</i><0.001 denotes significance between DSS treatment versus no treatment; # <i>P</i><0.05 denotes significance between Iron/DSS versus Ctrl/DSS or Iron versus Ctrl mice. Scale bar denotes 25 µm.</p

Hematology profile of Iron and Control mice treated with or without DSS.

<p>Results are expressed as mean±SEM; n = 7-8. * <i>P</i><0.05; denotes significance between DSS treatment versus no treatment and # <i>P</i><0.05; denotes significance between Iron/DSS versus Control/DSS or Iron versus Control mice. Hb, hemoglobin; MCH, mean cell hemoglobin; MCV, mean cell volume; RBC, red blood cell count; WBC, white blood cell count.</p

Dietary iron promoted colonic tumor development in inflammation-associated tumorigenesis.

<p>Colonoscopy images (A) depict tumor development in Iron/DSS and Control (Ctrl)/DSS mice at day 42. Arrows indicate colonic tumors. Tumors were classified as high-grade dysplasia by hematoxylin and eosin staining (B). Colonic tumor number (C) and tumor score (D) were determined in Iron and Control (Ctrl) mice treated with or without DSS at day 42. Results are expressed as mean±SEM, n = 6. * <i>P</i><0.05 denotes significance between DSS treatment versus no treatment; # <i>P</i><0.05 denotes significance between Iron/DSS versus Ctrl/DSS or Iron versus Ctrl mice. Scale bar denotes 100 µm.</p

Dietary iron enhances colonic inflammation and IL-6/IL-11-Stat3 signaling promoting colonic tumor development in mice

Chronic intestinal inflammation and high dietary iron are associated with colorectal cancer development. The role of Stat3 activation in iron-induced colonic inflammation and tumorigenesis was investigated in a mouse model of inflammation-associated colorectal cancer. Mice, fed either an iron-supplemented or control diet, were treated with azoxymethane and dextran sodium sulfate (DSS). Intestinal inflammation and tumor development were assessed by endoscopy and histology, gene expression by real-time PCR, Stat3 phosphorylation by immunoblot, cytokines by ELISA and apoptosis by TUNEL assay. Colonic inflammation was more severe in mice fed an iron-supplemented compared with a control diet one week post-DSS treatment, with enhanced colonic IL-6 and IL-11 release and Stat3 phosphorylation. Both IL-6 and ferritin, the iron storage protein, co-localized with macrophages suggesting iron may act directly on IL-6 producing-macrophages. Iron increased DSS-induced colonic epithelial cell proliferation and apoptosis consistent with enhanced mucosal damage. DSS-treated mice developed anemia that was not alleviated by dietary iron supplementation. Six weeks post-DSS treatment, iron-supplemented mice developed more and larger colonic tumors compared with control mice. Intratumoral IL-6 and IL-11 expression increased in DSS-treated mice and IL-6, and possibly IL-11, were enhanced by dietary iron. Gene expression of iron importers, divalent metal transporter 1 and transferrin receptor 1, increased and iron exporter, ferroportin, decreased in colonic tumors suggesting increased iron uptake. Dietary iron and colonic inflammation synergistically activated colonic IL-6/IL-11-Stat3 signaling promoting tumorigenesis. Oral iron therapy may be detrimental in inflammatory bowel disease since it may exacerbate colonic inflammation and increase colorectal cancer risk