James D. Bortner

Proteomic profiling of human plasma by iTRAQ reveals down-regulation of ITI-HC3 and VDBP by cigarette smoking.

Biomarkers in noninvasive fluids indicative of cigarette smokes effects are urgently needed. In this pilot study, we utilized the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to identify differentially expressed plasma proteins in healthy cigarette smokers compared to healthy nonsmokers; select proteins were further confirmed by immunoblot analysis. Significant, differentially expressed proteins identified in the plasma separated subjects based on their condition as smokers or nonsmokers. Several of the proteins identified in this study are associated with immunity and inflammatory responses and have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Proteins up-regulated in smokers included complement component 8 polypeptide chains α, β, and γ, and mannose-binding protein C, and proteins down-regulated included inter-α-trypsin inhibitor heavy chain H3 (ITI-HC3) and vitamin D-binding protein (VDBP). In addition, gelsolin and vitronectin, known tissue leakage proteins, were up- and down-regulated, respectively. Our results demonstrate for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome. Proteins identified in this pilot study may serve as candidate biomarkers of diseases resulting from exposure to cigarette smoke in future molecular epidemiological studies.

Modeling inflammation and oxidative stress in gastrointestinal disease development using novel organotypic culture systems

Gastroesophageal reflux disease (GERD), Barretts esophagus (BE), graft-versus-host disease (GVHD), and inflammatory bowel diseases such as ulcerative colitis and Crohns disease are common human gastrointestinal diseases that share inflammation as a key driver for their development. A general outcome resulting from these chronic inflammatory conditions is increased oxidative stress. Oxidative stress is caused by the generation of reactive oxygen and nitrogen species that are part of the normal inflammatory response, but are also capable of damaging cellular DNA, protein, and organelles. Damage to DNA can include DNA strand breaks, point mutations due to DNA adducts, as well as alterations in methylation patterns leading to activation of oncogenes or inactivation of tumor suppressors. There are a number of significant long-term consequences associated with chronic oxidative stress, most notably cancer. Infiltrating immune cells and stromal components of tissue including fibroblasts contribute to dynamic changes occurring in tissue related to disease development. Immune cells can potentiate oxidative stress, and fibroblasts have the capacity to contribute to advanced growth and proliferation of the epithelium and any resultant cancers. Disease models for GERD, BE, GVHD, and ulcerative colitis based on three-dimensional human cell and tissue culture systems that recapitulate in vivo growth and differentiation in inflammatory-associated microphysiological environments would enhance our understanding of disease progression and improve our ability to test for disease-prevention strategies. The development of physiologically relevant, human cell-based culture systems is therefore a major focus of our research. These novel models will be of enormous value, allowing us to test hypotheses and advance our understanding of these disorders, and will have a translational impact allowing us to more rapidly develop therapeutic and chemopreventive agents. In summary, this work to develop advanced human cell-based models of inflammatory conditions will greatly improve our ability to study, prevent, and treat GERD, BE, GVHD, and inflammatory bowel disease. The work will also foster the development of novel therapeutic and preventive strategies that will improve patient care for these important clinical conditions.

Down-regulation of 14-3-3 isoforms and annexin A5 proteins in lung adenocarcinoma induced by the tobacco-specific nitrosamine NNK in the A/J mouse revealed by proteomic analysis.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in the A/J mouse model. Here we identified and validated, using two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry and immunoblotting, proteins that are differentially expressed in the lungs of mice treated with NNK versus vehicle control treatment. We also determined whether protein levels in the lungs of NNK-treated mice could be further modulated by the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC). The proteins identified in this study are SEC14-like 3, dihydropyrimidinase-like 2, proteasome subunit alpha type 5, annexin A5, 14-3-3 protein isoforms (theta, epsilon, sigma, and zeta), Rho GDP dissociation inhibitor alpha, myosin light polypeptide 6, tubulin-alpha-1, vimentin, Atp5b protein, alpha-1-antitrypsin, and Clara cell 10 kDa protein (CC10). Among those proteins, we demonstrated for the first time that 14-3-3 isoforms (theta, epsilon, and sigma) and annexin A5 were significantly down-regulated in mouse lung adenocarcinoma induced by NNK and were recovered by p-XSC. These proteins are involved in a variety of biological functions that are critical in lung carcinogenesis. Identification of these proteins in surrogate tissue in future studies would be highly useful in early detection of lung adenocarcinoma and clinical chemoprevention trials.

Modeling human gastrointestinal inflammatory diseases using microphysiological culture systems

Gastrointestinal illnesses are a significant health burden for the US population, with 40 million office visits each year for gastrointestinal complaints and nearly 250,000 deaths. Acute and chronic inflammations are a common element of many gastrointestinal diseases. Inflammatory processes may be initiated by a chemical injury (acid reflux in the esophagus), an infectious agent (Helicobacter pylori infection in the stomach), autoimmune processes (graft versus host disease after bone marrow transplantation), or idiopathic (as in the case of inflammatory bowel diseases). Inflammation in these settings can contribute to acute complaints (pain, bleeding, obstruction, and diarrhea) as well as chronic sequelae including strictures and cancer. Research into the pathophysiology of these conditions has been limited by the availability of primary human tissues or appropriate animal models that attempt to physiologically model the human disease. With the many recent advances in tissue engineering and primary human cell culture systems, it is conceivable that these approaches can be adapted to develop novel human ex vivo systems that incorporate many human cell types to recapitulate in vivo growth and differentiation in inflammatory microphysiological environments. Such an advance in technology would improve our understanding of human disease progression and enhance our ability to test for disease prevention strategies and novel therapeutics. We will review current models for the inflammatory and immunological aspects of Barretts esophagus, acute graft versus host disease, and inflammatory bowel disease and explore recent advances in culture methodologies that make these novel microphysiological research systems possible.

Modulations of benzo[a]pyrene-induced DNA adduct, cyclin D1 and PCNA in oral tissue by 1,4-phenylenebis(methylene)selenocyanate

Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.

Proteomic Changes Induced by Effective Chemopreventive Ratios of n-3:n-6 Fatty Acids and Tamoxifen against MNU-Induced Mammary Cancer in the Rat

We used a proteomic approach to gain insights into the mechanisms of protection at the protein level by a high n-3:n-6 ratio in the absence and presence of Tamoxifen. Four groups were treated with 1-methyl-1-nitrosourea (MNU) and fed the following diets with varied n-3:n-6 ratios; group 1 = 1:1; groups 2 and 3 = 10:1 and 25:1, respectively; group 4: (25:1) plus Tamoxifen (1 mg/kg diet). The plasma from six rats/group was pooled and analyzed with the isobaric tags for relative and absolute quantitation method; 148 proteins were identified with 95% confidence by ProteinPilot 4.0. In plasma of rats fed 10:1, 25:1 n-3:n-6, and 25:1 plus Tamoxifen, the number of proteins that met our criteria (P ≤ 0.05, error factor ≤ 2) were 10, 14, and 19 proteins, respectively. Selected proteins were further validated by Western blotting. Compared to 1:1, both 10:1 and 25:1 diets upregulated vitamin D binding protein, gelsolin, and 14-3-3 sigma, reported to have tumor suppressive effects, whereas alpha-1B-glycoprotein, which has been reported to be elevated in the serum of breast cancer patients was decreased. Compared to 25:1, the 25:1 plus Tamoxifen diet downregulated apolipoprotein E, haptoglobin, and inter-α-inhibitor H4 heavy chain. Ingenuity pathway analysis determined that the trends of specific proteins were related to lipid metabolism in the 25:1 n-3:n-6 group, whereas the 25:1 n-3:n-6 plus Tamoxifen group included proteins involved in cancer and inflammation. Our results show that several proteins were altered in a manner consistent with chemoprevention. Such proteins may serve as biomarkers to monitor efficacy of n-3 and Tamoxifen in future clinical chemoprevention trials. Cancer Prev Res; 6(9); 979–88. ©2013 AACR.

Sa1838 CDx2 Expression Reduces Disease Progression in the L2-IL-1Beta Mouse Model for Barrett's Esophagus

G A A b st ra ct s 3 intensity levels (walking, moderate intensity and vigorous intensity) during the past 7 days. We categorized level of physical activity by low, moderate, or high using IPAQ definitions. We also estimated metabolic equivalent minutes per week (MET-min/wk) by weighting the reported minutes/week within each activity category by a MET energy expenditure estimate for each category of activity. We calculated odds ratios (OR) and 95% confidence intervals (95% CI) using multivariable logistic regression models adjusting for age, sex, race, GERD symptoms, H. pylori, BMI and high waist-to-hip ratio. Results: There were 323 cases with BE and 1849 controls (1347 from endoscopy and 502 from the primary care clinic) (Table 1). Most (68.8%) patients were in the lowest category of physical activity, 13.5% had moderate activity and 11.2% had high activity (6.5% were missing). BE cases were more likely to be in the high category physical activity category than controls (13.6% vs. 10.8% p=0.08). The overall average MET-min/week for walking were 909 for BE cases vs. 561 in controls (p=0.16); with similar findings for those with moderate activity (1094 METmin/week for BE cases vs. 755 for controls, p=0.175), and for vigorous activity (783.8 METmin/week for BE cases vs. 826.2 for controls, p=0.927). In multivariable logistic regression, physical activity was not significantly associated with BE (OR for high vs. low physical activity: 1.19 (95%CI: 0.8-1.73). In fact, BE patients were more likely to have high level of physical activity than PCP controls (OR: 2.1; 95% CI: 1.17-3.6). Conclusions: Recent amount and intensity of physical activity does not seem to be associated with significant changes in the risk of BE. Studies are required examine long term effects of physical activity.

Abstract 670: Cigarette smoking, race, and genetic ancestry in association with plasma vitamin D-binding protein (VDBP) in healthy African Americans (AA) and Caucasian Americans (CA)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Vitamin D and VDBP affect several pathways involved in inflammation, tumor growth, and immune surveillance relevant to carcinogenesis. In a previous pilot study (Bortner et al., 2010), using a proteomics approach, VDBP was down-regulated in the plasma of chronic cigarette smokers by as much as 3-fold. Therefore, using an existing study of healthy AA and CA participants, we conducted an investigation of the differences in VDBP between 39 current smokers and 82 never smokers, frequency matched on age, sex and race/ethnicity. VDBP plasma concentrations were determined in duplicate using the Quantikine® Human VDBP ELISA (CV%, 5.6%). Genotyping was conducted for a set of 112 previously validated Ancestry Informative Markers for the percent West African and European genetic ancestry, and VDBP coding region changes via rs7041 (Asp416Glu, T/G) and rs4588 (Thr420Lys, C/A). Vitamin D metabolites (25OHD3 and 24,25(OH)2D3) were determined using LC-MS/MS (CV% 2.7 and 6.3, respectively). T-test, Chi-Square, Fishers Exact, and Spearmans correlation coefficients were used to determine statistically significant bivariate differences. Multiple logistic regression was used to adjust for multiple factors using the log-normal transformation of VDBP concentration with the Cochran-Armitage test for trend for VDBP genotype. Multiplicative interaction terms were tested along with their main-effects, adjusted for co-variates. There were no significant differences between current smokers and never smokers by age, VDBP genotype, body mass index, vitamin D metabolites, or genetic ancestry. VDBP concentrations were significantly negatively correlated with body mass index (BMI, r=−0.19, p=0.033), West African ancestry (r=−0.66, p<0.001), vitamin D metabolites (p-values<0.001), VDBP rs4588-A and rs7041-T alleles (VDBP genotypes were 100% correlated). VDBP genotypes coding Asp416Asp and Lys420Lys were at least 3.3-fold lower, compared with Glu416Glu and Thr420Thr, respectively, and this was regardless of smoking status (p-trend<0.001). There was no association between VDBP concentration and age or smoking status. In multiple regression models, serum vitamin D metabolites, self-reported race/ethnicity, West African ancestry and GC genotypes remained significantly associated with VDBP concentration. The magnitude of difference in these variables was similar among current and never smokers, although BMI remained significantly negatively associated with VDBP concentrations among never smokers (p-interaction =0.028). Smoking status was not highly correlated with VDBP plasma concentration, although may have some interactive effect with BMI. Future studies will need to account for the strong correlations with VDBP genotype and genetic ancestry. Support: 1K22CA120092-01A2 and 3K22CA120092-02S1. 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 670. doi:1538-7445.AM2012-670

Abstract 4572: Comparison of proteomic profiles in human plasma of smokers and nonsmokers using the iTRAQ approach

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is a potentially preventable disease because it is strongly related to tobacco smoke. Although there have been great strides taken to inform the population of the severity of the risks associated with smoking, there are currently 43.4 million Americans that smoke and 1.3 billion smokers worldwide. Therefore, it is important to develop biomarkers that can identify individual smokers and ex-smokers who are at especially high risk for lung cancer. Relative changes that take place in the human plasma proteome as a result of smoking may be playing an important role in the etiology of lung cancer. Thus, in this study we used a proteomic approach utilizing isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to compare the plasma protein expression levels between healthy smokers and non-smokers. With this multi-plex non-gel based technique, up to 8 samples can be simultaneously labeled with isobaric tagging reagents, 113-119 and 121, for quantitation and identification by mass spectrometry. Plasma samples from seven white male smokers and seven white male non-smokers were distributed into two 8-plex iTRAQ sets. The 113 label in each set was used to label a pool consisting of each sample in the study in order to compare between the two 8-plex sets. Plasma from each subject was depleted of the 14 most abundant plasma proteins using an immunoaffinity column to search for lower abundant proteins. Based on our results, we identified 18 significant (p<0.05) differentially expressed proteins between smokers and non-smokers. The proteins that were down-regulated in smokers include sex hormone-binding globulin (SHBG), inter-α (globulin) inhibitor H3 (HC3), and vitronectin. The proteins that were up-regulated in smokers include the complement component 8, α, β and γ polypeptides, gelsolin, and mannose-binding protein C (MBP-C). Among those identified, SHBG, HC3, and gelsolin protein have been previously identified in smokers and have been suggested to be candidate markers for lung cancer. The results of this study show that cigarette smoking can influence the relative expression profile of plasma proteins. However, the relationship of these proteins to the development of lung cancer needs to be determined so that they can be further developed as biomarkers of disease risk. Support: NCI PO1 70972 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4572.

Abstract 4589: Changes in proteomic profiles with age in male rats using the iTRAQ approach

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aging is an important risk factor for many diseases including prostate cancer (PC). Enhanced risk for disease may, in part, be due to aging-related changes in the proteome. Thus, a better knowledge of the changes in protein levels occurring with age would help in the early detection of the disease, as well as in monitoring chemopreventive intervention. In the present study we used male F344 rats, a commonly used aging and PC model, and compared protein levels in the blood plasma of young (4 months), mature (12 months), old (18 months) and very old (24 months) rats by a proteomic approach known as isobaric Tag for Relative and Absolute Quantitation (iTRAQ). In this multi-plex non-gel based technique samples can be simultaneously labeled with isobaric tagging reagents for quantitation and identification by mass spectrometry. Plasma samples were pooled from three animals and 50 μg of protein from each group was labeled for a 4-plex iTRAQ analysis. A total of seventy nine proteins were identified, thirty one with >99% confidence. Among those, the levels of thirteen proteins were changed significantly (p 99% confidence including serotransferrin, hemopexin, fetuin-A, fetuin-B and α-1-acid glycoprotein. Both age-specific and growth-development related changes were observed throughout the life span with maximum changes (either increases or decreases) being observed at 18 months. The results of this study show that age indeed can influence the expression profile of plasma proteins. The relationship of these proteins to PC is currently being investigated by analyzing their levels in different lobes of the prostate gland. Supported in part by R01 CA 127729-01 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4589.