Michael W. Schäffer

Identification of lutein, a dietary antioxidant carotenoid in guinea pig tissues.

Lutein, a dietary carotenoid, is a well known antioxidant. The major source of this carotenoid in humans is diet. We report here the presence of lutein, a dietary carotenoid in several guinea pig tissues (in decreasing order: liver>spleen>lung>>testis>kidney>plasma>eye but not in white adipose tissue). The presence of lutein in lung and other tissues may be significant in term of its antioxidant capacity of these organs.

Implications of the colonic deposition of free hemoglobin-α chain: a previously unknown tissue by-product in inflammatory bowel disease.

Background:We analyzed inflamed mucosal/submucosal layers of ulcerative colitis (UC = 63) and Crohns colitis (CC = 50), and unexpectedly, we unveiled a pool of free hemoglobin alpha (Hb-&agr;) 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-&agr; is key transformational stepping that increases colitis-related colorectal cancer vulnerability. Methods: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 = &agr;, &bgr;, 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 &mgr;M and 100 &mgr;M of Hb(n)/Hb, individually for 2, 6, and 12 hours. Quantification of Hb-&agr; 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 &bgr;-actin. Results:Immunohistochemistry of CC/UC mucosal/submucosal compartments stained strongly positive for Hb-&agr; and significantly higher versus Ctrl. NCM460 exposed to Hb(n)/Hb exhibited steadily increasing ROS and subsequent DNAD. DNAD was higher in 10 &mgr;M than 100 &mgr;M in Hb-&bgr;/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 (P < 0.05). 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 (P < 0.01). Conclusions:In the colitides, mucosal/submucosal tissue microenvironments demonstrated pool of free Hb-&agr; chain. In vitro exposure of NCM460 cells to Hb(n)/Hb induced ROS and DNAD. Toxic effect of free Hb-&agr;, 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.

Uptake of all-trans retinoic acid-containing aerosol by inhalation to lungs in a guinea pig model system—A pilot study

ABSTRACT Systemic therapies with retinoic acid (RA) can result in toxic side effects without yielding biologically effective levels in target tissues such as lung. The authors adapted a PARI LC Star nebulizer to create a tubular system for short-term inhalation treatment of guinea pigs using a water-miscible formulation of all-trans RA (ATRA) or vehicle. Based on the initial average weight, animals received an estimated average ATRA doses of either 0.32 mg·kg−1 (low dose, 1.4 mM), or 0.62 mg·kg−1 (medium dose, 2.8 mM), or 1.26 mg·kg−1 (high dose, 5.6 mM) 20 minutes per day for 6 consecutive days. This system led to a rise of ATRA levels in lung, but not liver or plasma. Cellular lung levels of retinol, retinyl palmitate, and retinyl stearate also appeared to be unaffected (245.6 ± 10.7, 47.4 ± 3.4, and 132.8 ± 7.7 ng·g−1 wet weight, respectively). The application of this aerosolized ATRA also induced a dose-dependent protein expression of the cellular retinol-binding protein 1 (CRBP-1) in lung, without apparent harmful side effects.

Abstract 2058: Colorectal cancer in the setting of inflammatory bowel disease: role of hemoglobin

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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 Crohns 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

Abstract LB-428: Upregulation of serum amyloid A1 (SAA 1) gene is associated with ulcerative colitis-related colon cancer

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), Crohn9s 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

Abstract LB-450: Gene expression of colonic submucosa differs between the inflammatory colitides. A possible reason for differences in IBD-associated CRC incidences

Purpose : Differentiating ulcerative colitis (UC) and Crohn9s 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

Abstract LB-463: Proteomic patterns of colonic submucosa delineates the inflammatory colitides. This could aid understand IBD-related colorectal malignancy

Purpose : Differentiating Crohn9s 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 ( m/z ) values between normal vs the IBD and more strikingly between CC vs. UC (p Conclusions : MALDI-MS tissue profiling as described distinguished the colitides. The methodology revealed 5 m/z peaks of interest. Analyses are underway to identify these IBD discriminative proteins. We hypothesize that these biomarker candidates that distinguish UC from CC will allow delineation of indeterminate colitis into UC or CC. Whether these protein fingerprints play a role in colonic carcinogenesis remains to be elucidated. 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

Abstract A45: Molecular targets in early detection and differentiation of inflammatory bowel disease-associated colon-rectal-anal cancer disparities

The inflammatory bowel disease (IBD), Crohn9s (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 colorectalanal 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 disagreements, the goal is clear: 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. The results 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 segments 2,3 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. M9Koma 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. M9Koma 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, M9Koma AE. Pouch-related dysplasia and adenocarcinoma following restorative proctocolectomy for ulcerative colitis. Tech coloproctol 2011;15:7–16. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B33.