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In terms of their involvement in allergic and inflammatory conditions, mast cells (MC) can be promising targets for medical agents in therapy. Because of their good compliance and effectiveness, phytochemicals are of great interest as new therapeutic tools in form of nutraceuticals. We found recently that cinnamon extract (CE) inhibits mast cell activation. Here, we analysed the effects of a major compound of CE, cinnamaldehyde (CA), on mast cell activation. Release of prestored and de novo synthesised mediators as well as expression of pro-inflammatory cytokines and mast cell-specific proteases were analysed in RBL-2H3 cells or in human mast cells isolated from intestinal tissue (hiMC) treated with CA prior to stimulation by FceRI crosslinking or IONO/PMA. The results were compared with the corresponding effects of CE. Following treatment with CA, release of β-hexosaminidase in IgE-dependent or IgE-independent activated RBL-2H3 cells was down-regulated in a dose-dependent manner to about 10 %. In hiMC, release of β-hexosaminidase was also significantly reduced, and release of LTC4 and CXCL8 was almost completely inhibited by CA. Moreover, IgE-mediated expression of CXCL8, CCL2, CCL3 and CCL4 in hiMC was significantly down-regulated by CA. With the exception of the expression of the mast cell proteases tryptase and chymase, the inhibitory effects of CA were very similar to the effects shown for CE treatment. The reducing effect of CA on mast cell mediators—seen for long- and for short-term incubations—could be related to particular signalling pathways as CA caused a down-regulation in ERK as well as PLCγ1 phosphorylation. CA decreases release and expression of pro-inflammatory mast cell mediators. This inhibitory action is similar to the effects observed for CE indicating CA as the main active compound in CE leading to its anti-allergic properties.

We analyzed inflamed mucosal/submucosal layers of ulcerative colitis (UC = 63) and Crohn's colitis (CC = 50), and unexpectedly, we unveiled a pool of free hemoglobin alpha (Hb-α) chain. Patients with colitides have increased reactive oxidative stress (ROS), DNA oxidation products, free iron in mucosa, in preneoplastic, and in colitis-cancers and increased risks of developing colorectal cancer. All inflammatory bowel disease-related colorectal cancer lesions are found in segments with colitis. Linking this information, we investigated whether free Hb-α is key transformational stepping that increases colitis-related colorectal cancer vulnerability.UC/CC samples were profiled using matrix-assisted laser desorption/ionization mass spectrometry; protein identification was made by liquid chromatography. Diverticulitis was used as control (Ctrl). The presence of Hb(n) (n = α, β, or hemin)/Hb was validated by Western blotting and immunohistochemistry. We tested for DNA damage (DNAD) by exposing normal colonic epithelial cell line, NCM460, to 10 μM and 100 μM of Hb(n)/Hb, individually for 2, 6, and 12 hours. Quantification of Hb-α staining was done by Nikon Elements Advance Research Analysis software. ROS was measured by the production of 8-OHdG. DNAD was assessed by Comet assay. Colonic tissue homogenate antioxidants Nrf2-, CAT-, SOD-, and GPx-expressions were analyzed densitometrically/normalized by β-actin.Immunohistochemistry of CC/UC mucosal/submucosal compartments stained strongly positive for Hb-α and significantly higher versus Ctrl. NCM460 exposed to Hb(n)/Hb exhibited steadily increasing ROS and subsequent DNAD. DNAD was higher in 10 μM than 100 μM in Hb-β/hemin the first 2 hours then plateaued followed by DNAD repair. This may be likely due to apoptosis in the later concentration. Nrf2 enzyme activities among UC, CC, and ulcerative colitis-associated colon cancer (UCAC) were observed impaired in all inflammatory bowel disease subjects. Decreased levels of Nrf2 among patients with UC versus patients with CC with active disease were insignificant as well as versus Ctrls but significantly lower in UCAC versus Ctrl. SOD was decreased in UC and UCAC and GPx in CC but statistically not significant. Comparing CC versus UC, SOD was significantly lower in CC (P0.05). CAT was observed increased among patients with CC/UC/UCAC and GPx in UC and UCAC versus Ctrl, respectively, and significantly increased in CC versus Ctrl (P0.01).In the colitides, mucosal/submucosal tissue microenvironments demonstrated pool of free Hb-α chain. In vitro exposure of NCM460 cells to Hb(n)/Hb induced ROS and DNAD. Toxic effect of free Hb-α, in colonic epithelial cells, is therefore through production of ROS formation modulated by impairment of antioxidant effects. Targeting reduction-oxidation-sensitive pathways and transcription factors may offer options for inflammatory bowel disease-management and colitis-related cancer prevention.

The incidence of familial adenomatous polyposis (FAP) is one in 7,000 to 12,000 live births. Virtually, all surgically untreated patients with FAP inevitably develop colorectal-cancer in their lifetime because they carry the adenomatous polyposis coli gene. Thus prophylactic proctocolectomy is indicated. Surgical treatment of FAP is still controversial. There are however, four surgical options: ileorectal anastomosis, restorative proctocolectomy with ileal pouch-anal anastomosis, proctocolectomy with ileostomy, and proctocolectomy with continent-ileostomy. Conventional proctocolectomy options largely lie between colectomy with ileorectal anastomosis or ileal pouch-anal anastomosis. Detractors of ileal pouch-anal anastomosis prefer ileorectal anastomosis because of better functional results and quality of life. The functional outcome of total colectomy with ileorectal anastomosis is undoubtedly far superior to that of the ileoanal pouch; however, the risk for rectal cancer is increased by 30%. Even after mucosectomy, inadvertent small mucosal residual islands remain. These residual islands carry the potential for the development of subsequent malignancy. We reviewed the literature (1975-2012) on the incidence, nature, and possible etiology of subsequent ileal-pouch and anal transit zone adenocarcinoma after prophylactic surgery procedure for FAP. To date there are 24 studies reporting 92 pouch-related cancers; 15 case reports, 4 prospective and 5 retrospective studies. Twenty three of 92 cancers (25%) developed in the pouch mucosa and 69 (75%) in anal transit zone (ATZ). Current recommendation for pouch surveillance and treatment are presented. Data suggest lifetime surveillance of these patients.

Vitamin A (VA) and its active metabolites play an essential role in lung airway function. Patients with moderate to severe chronic obstructive pulmonary disease (COPD) have a lower serum retinol concentration, and improvement of their 1-second Forced Expiratory Volume (FEV1) is achieved with VA supplementation. In order to test our hypothesis that the VA signaling pathway is compromised in COPD, we obtained 20 lung samples from COPD patients differing in the degree of emphysema as judged by their FEV% values. All were smokers or were exposed to secondhand smoke. Levels of VA (retinol/retinyl ester), tocopherols and carotenoids (lutein, beta-carotene) in these samples were determined using HPLC. Additional analytes beside VA were included for their known roles as antioxidants and modulators of VA-action. VA levels (retinol/retinyl ester) decreased significantly with the increase in severity of emphysema. Among other analytes, α-tocopherol levels fell by 25.8% in the severe emphysema group in comparison to the mild emphysema group, and lutein levels similarly decreased in severe compared to moderate emphysema groups. However, beta-carotene levels remained unchanged. Thus there is a significant linear correlation between lung VA-levels and the severity of emphysema. There was also a significant reduction in the levels of α-, δ-tocopherol and lutein in the severe emphysema group of COPD patients who either smoked or were exposed to smoke.

Allergic diseases with mast cells (MC) as main effector cells show an increased prevalence. MC also play an essential role in other inflammatory conditions. Therapeutical use of anti-inflammatory nutraceuticals directly targeting MC activation could be of interest for afflicted patients. Nobiletin and tangeretin are citrus peel polymethoxyflavones, a group of citrus flavonoids, possessing anticancer, antimetastatic, and anti-inflammatory activities. Here, we analyzed the effects of nobiletin/tangeretin on LPS- and IgE-mediated stimulation of human intestinal mast cells (hiMC). MC isolated from human intestinal tissue were treated with different concentrations of nobiletin or tangeretin prior to stimulation via LPS/sCD14 or IgE-dependently. Degranulation, pro-inflammatory cytokine expression and phosphorylation of ERK1/2 were examined. Expression of CXCL8, CCL3, CCL4 and IL-1β in response to LPS-mediated stimulation was inhibited by nobiletin/tangeretin. hiMC activated IgE-dependently showed a reduced release of β-hexosaminidase and cysteinyl LTC4 in response to nobiletin, but not in response to tangeretin. Expression of CXCL8, CCL2, CCL3, CCL4 and TNF in IgE-dependently activated hiMC was decreased in a dose-dependent manner following treatment with nobiletin/tangeretin. IL-1β expression was only reduced by tangeretin. Compared to treatment with NF-κB inhibitor BMS345541 or MEK-inhibitor PD98059, nobiletin and tangeretin showed similar effects on mediator production. Phosphorylation of ERK1/2 upon IgE-mediated antigen stimulation was significantly suppressed by nobiletin and tangeretin. Nobiletin and, to a lesser extent, tangeretin could be considered as anti-inflammatory nutraceuticals by reducing release and production of proinflammatory mediators in MC.

Colorectal carcinoma (CRC) is a serious complication of inflammatory bowel disease (IBD) and accounts for approximately 15% of all IBD-associated deaths. The likelihood of IBD-related carcinoma is greater than that of sporadic CRC. Over one half are diagnosed at stage III or IV. During the last few years we have worked on mining colon mucosal and submucosal layers for discovery of biomarkers that differentiate ulcerative colitis (UC) from Crohn's colitis (CC). Using LC-MS/MS, we examined signals found to be significantly different between CC and UC samples. We found a signal at m/z 5045 which was more intense in UC samples. The MALDI spectrum did not identify an intact protein entity but did identify hemoglobin chains. Macrophages are highly versatile phagocytes active in multiple roles in the immune system and key players in the inflammatory response. The microenvironment of most inflammation is filled with a large population of macrophages. In IBD, studies have found that macrophages can count for more than 50% of the exudative mass. Their presence within the inflammatory microenvironment, in some cases, has been proven to increase transformation, angiogenesis, and immunosuppression. In hemorrhagic situations (as in UC) macrophages engulf erythrocytes and as a result release free heme iron (heFe). Earlier studies observed that heFe has cellular proliferation effects on colon cancer cells. Recently, the potential carcinogenic effects of heFe were documented when it was shown that heFe increases the number of aberrant crypt foci in colon mucosa. In the colon, iron is expected to increase the production of reactive oxygen (O2) species (ROS) from peroxides via the Fenton reaction, which may be the cause of cellular toxicity and even pro-mutagenic lesions. Intracellular reactions with active O2 can result in the initiation and progression of carcinogenesis by induction of gene mutations, chromosomal damage and cytotoxic effects. We hypothesize that elevated expression of mucosal free heFe would be associated with an increased risk of UC-associated CRC. To validate this will require investigating whether hemoglobin could be classified as a proliferative or transforming agent for colon cancer cells by causing reactive oxygen species release. For this purpose, we plan to study the cellular viability of differentiated colon cell line (cancer: CCL 224, CCL 227 and normal: NCM 356 and NCM 460) after administration of hemoglobin at different concentrations. ROS production will be investigated in each step. Additionally, we intend to examine the potential cytotoxicity of hemoglobin. Supported: MMC-VICC Cancer Partnership Grant # 3U54CA091408-09S 1 (SEA & HLM); MeTRC grant # 5U54RR026140-03 (SEA), and Vanderbilt SPORE in GI Cancer Grant # P50CA095103 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2058. doi:1538-7445.AM2012-2058

The inflammatory bowel disease (IBD), Crohn's (CC) and ulcerative colitis (UC), affect approximately 1–2 of every 1000 people in developed countries. These chronic inflammatory diseases result in significant morbidity and mortality. All IBD-associated colorectal-anal cancers (CRAC) occurred in segments of colitis and are frequently diagnosed at an advanced stage. This presentation is a continuation of our work that investigates potential molecules that could define a unique classifier between CC and UC and early detection of CRAC. There are multiple challenges to identifying protein classifiers suggesting outcome prediction and differentiation for patients with IBD and/or IBD-CRAC from molecular interpretation standpoints is complex. While there are technical approach advances, the goal is clear however: to produce scientific evidence which can provide personalized expert care to patients. We have developed an amenable proteomic methodology that supports the diagnostic feasibility to discriminate molecularly, different inflammatory colitis. The histologic layers of colectomy samples from patients with confirmed UC and CC tissues were analyzed using matrix-assisted laser de-sorption/ionization mass spectrometry (MALDI MS) for proteomic profiling. Our previous findings (1) prompted further sample collection resulting in an increased sample size that would allow a more robust analysis. The samples from colon tissues collected in 2008 and samples collected in 2010 were re-randomized into training and independent test sets in order to avoid systematic differences between new and old data sets, typical for MALD-ToF spectra acquired in situ from tissue at different times. MALDI-ToF spectra were included in the analysis only for samples containing more than 3 unique spectra. Our studies have successfully identified 11 highly significant mass-to-charge ratio (m/z) signals (m/z 5045, 6139, 9245, 8413, 3666, 3595, 4122, 8774, 2778, 9232 and 9519) that distinguish CC from UC. These features are independent of the tissue of origin and represent disease specific markers. Some of these signatures were only found in the colonic mucosa (m/z 8413, 3666 & 3595) or submucosa (m/z 4122, 8774, 2778, 9232 & 9519) while others were found in both two layers (m/z 5045, 6139 & 9245). This information may provide new avenues for the development of novel diagnostic, prognostic and therapeutic targets. We will analyze CRAC in IBD segments2,3 to look for these proteins that may help in studying their biological mechanisms in cancer transformation. Support: 3U54CA091408-09S1 (to MMC-VICC Partnership: SE Adunyah & HL Moses) References: 1. M'Koma AE, Seely EH, Washington MK, Schwartz DA, Muldoon RL, Herline AJ, Wise PE, Caprioli RM Proteomic Profiling of Mucosal and Submucosal Colonic Tissues Yields Protein Signatures that Differentiate the Inflammatory Colitides. Inflamm Bowel Dis 2011;17:875-83. 2. M'Koma AE, Moses HL, Adunyah SE. Inflammatory bowel disease-associated colorectal cancer: proctocolectomy andmucosectomy does not necessarily eliminate pouch related cancer incidences. Int J colorect Dis 2011;26:533-52. 3. Um JW, M'Koma AE. Pouch-related dysplasia and adenocarcinoma following restorative proctocolectomy for ulcerative colitis. Tech coloproctol 2011;15:7-16. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A45.

Purpose: Differentiating ulcerative colitis (UC) and Crohn's colitis (CC) may be inaccurate in up to 30% of inflammatory bowel disease (IBD) cases due to overlapping features of these colitides. IBD-associated colorectal cancer (CRC) is frequently diagnosed in an advanced stage. Mass-spectrometric (MS) proteomic patterns found in colonic submucosa have been shown to discriminate UC and CC. To verify this, we aimed to conduct pilot gene expression analyses via microarray assessment of colonic submucosa. The significances of these genes to CRC initiation is as yet not been elucidated. Methods: Laser capture microdissection of colonic submucosa from UC (n=8), CC (n=8), and normal (NL, n=8) samples was performed. The submucosal mRNA was extracted using the PicoPure(TM) RNA Isolation Kit. Comprehensive gene expression analysis of the pooled mRNA from each group was then performed using the Affymetrix GeneChip® Gene 1.0 ST Array System. To detect changes for UC, we compared UC to CC and UC to NL; and for CC, we compared CC to UC and CC to NL. Results: 28,869 genes were represented on the array by 26 probes spread across the full length of each gene. When comparing UC to CC, 138 genes showed at least a 5-fold significant overexpression (p Conclusions: Microarray assessments show significantly different gene overexpression in UC and CC colonic submucosa, with a few select genes showing dramatic overexpression. Preliminary results indicate that we have the tools in hand to successfully validate the results from the microarray analysis using TR-qPCR and immunohistochemistry (IHC). These genes may assist in delineation of the previously identified differentiating MS submucosal proteomic peaks, potentially facilitating protein biomarker identification to discriminate the inflammatory colitides. These genes may relate to CRC initiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-450. doi:10.1158/1538-7445.AM2011-LB-450

Purpose: Differentiating Crohn's colitis (CC) and ulcerative colitis (UC) can be challenging even in combination of clinical, endoscopic, radiologic and histopathology examination. Biomarker studies have thus far been unsuccessful for disease delineation. We aim to use unique tissue proteomic methods to evaluate colonic tissue layers for potential biomarkers to identify CC vs. UC. Methods: Fresh-frozen colon specimens from resections for IBD and/or colorectal cancer were retrospectively retrieved. Colitis diagnoses were histologically re-confirmed by a blinded gastrointestinal pathologist. Three sample groups (n=5 each group) were examined: normal colon from CRC specimens (control), UC & CC. MALDI-MS was used to profile mucosal and submucosal compartments individually. Frozen tissues were sectioned at ∼10–15 μm for mounting onto either metal or conductive glass target plates (the glass plates allowing for histologic and MALDI-MS analysis on the same section). Sinapinic acid (20 mg/mL in 50:50 acetonitrile: 0.1% TFA in water) was used to give the best combination of uniform crystal coverage and signal quality for direct tissue protein analysis. Results: MALDI-MS achieved high mass accuracy (±0.01 Daltons) in the lower mass range ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-463. doi:10.1158/1538-7445.AM2011-LB-463

Purpose: Ulcerative colitis (UC)-associated colon cancer is frequently diagnosed in an advanced stage. SAA1 is a secreted protein made in the liver and circulates in low levels in blood. SAA1 gene is expressed in colon carcinomas and is a chemoattractant with induction of migration, adhesion, and tissue infiltration of monocytes and Polymorphnuclear-leucocytes (PMN-L). Methods: Laser capture microdissection of colonic submucosa from UC (n=8), Crohn's colitis (CC) (n=8)), and normal (NL, n=8) samples was performed. The submucosal mRNA was extracted using the PicoPure(TM) RNA Isolation Kit. Comprehensive gene expression analysis of the pooled mRNA from each group was then performed using the Affymetrix GeneChip® Gene 1.0 ST Array System. Statistical comparisons of UC vs. CC and NL as well as CC versus UC and NL were performed (Wilcoxon Rank Test). To detect changes for UC, we compared UC to CC and UC to NL; and for CC, we compared CC to UC and CC to NL. Results: Analysis of all UC vs. CC and NL controls showed 28,869 genes which were represented on the array by 26 probes spread across the full length of each gene. When compared UC vs. CC and NL SAA1 remained the most upregulated gene showing a 56.8-folds and 91.3-folds (p< 0.0001) over expression. Conclusion: Microarray assessments show significantly overexpression of SAA1 in UC specimens. SAA1 has a role in local inflammation in the microenvironment of malignant tissue and is expressed in colon carcinomas. Our finding may potentially facilitate and validate the SAA1 as a new early predictive clinical marker and a unique target for designing novel selective inhibitors for therapeutic intervention of UC-associated CRC. Acknowledgement: 3U54CA091408–09S1 (MMC-VICC partnership) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-428. doi:10.1158/1538-7445.AM2011-LB-428