Jeremy N. Myers

In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells.

The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628bp fragment (-613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site of SLUG. SLUG specifically inhibited VDR gene promoter activity. Chromatin-immunoprecipitation (ChIP) assays revealed that SLUG is recruited on the native VDR gene promoter along with the co-repressor protein CtBP1 and the effector protein HDAC1. These data suggests that SLUG binds to the E2-box sequences of the VDR gene promoter and recruits CtBP1 and HDAC1, which results in the inhibition of VDR gene expression by chromatin remodeling.

Chemoprevention of benzo(a)pyrene-induced colon polyps in ApcMin mice by resveratrol

Human dietary exposure to benzo(a)pyrene (BaP) has generated interest with regard to the association of BaP with gastrointestinal carcinogenesis. Since colon cancer ranks third among cancer-related mortalities, it is necessary to evaluate the effect of phytochemicals on colon cancer initiation and progression. In this study, we investigated the preventive effects of resveratrol (RVT) on BaP-induced colon carcinogenesis in Apc(Min) mouse model. For the first group of mice, 100 μg BaP/kg body weight was administered to mice in peanut oil via oral gavage over a 60-day period. For the second group, RVT was coadministered with BaP at a dose of 45 μg/kg. For the third group, RVT was administered for 1 week prior to BaP exposure for 60 days. Jejunum, colon and liver were collected at 60 days post BaP and RVT exposure; adenomas in jejunum and colon were counted and subjected to histopathology. RVT reduced the number of colon adenomas in BaP+RVT-treated mice significantly compared to that in mice that received BaP alone. While dysplasia of varying degrees was noted in colon of BaP-treated mice, the dysplasias were of limited occurrence in RVT-treated mice. To ascertain whether the tumor inhibition is a result of altered BaP-induced toxicity of tumor cells, growth, apoptosis and proliferation of adenocarcinoma cells were assessed posttreatment with RVT and BaP. Cotreatment with RVT increased apoptosis and decreased cell proliferation to a greater extent than with BaP alone. Overall, our observations reveal that RVT inhibits colon tumorigenesis when given together with BaP and holds promise as a therapeutic agent.

Influence of dietary fat type on benzo(a)pyrene [B(a)P] biotransformation in a B(a)P-induced mouse model of colon cancer.

In the US alone, around 60,000 lives/year are lost due to colon cancer. Diet and environment have been implicated in the development of sporadic colon tumors. The objective of this study was to determine how dietary fat potentiates the development of colon tumors through altered B(a)P biotransformation, using the Adenomatous polyposis coli with Multiple intestinal neoplasia mouse model. Benzo(a)pyrene was administered to mice through tricaprylin, and unsaturated (USF; peanut oil) and saturated (SF; coconut oil) fats at doses of 50 and 100 μg/kg via oral gavage over a 60-day period. Blood, colon, and liver were collected at the end of exposure period. The expression of B(a)P biotransformation enzymes [cytochrome P450 (CYP)1A1, CYP1B1 and glutathione-S-transferase] in liver and colon were assayed at the level of protein, mRNA and activities. Plasma and tissue samples were analyzed by reverse phase high-performance liquid chromatography for B(a)P metabolites. Additionally, DNA isolated from colon and liver tissues was analyzed for B(a)P-induced DNA adducts by the (32)P-postlabeling method using a thin-layer chromatography system. Benzo(a)pyrene exposure through dietary fat altered its metabolic fate in a dose-dependent manner, with 100 μg/kg dose group registering an elevated expression of B(a)P biotransformation enzymes, and greater concentration of B(a)P metabolites, compared to the 50 μg/kg dose group (P<.05). This effect was more pronounced for SF group compared to USF group (P<.05). These findings establish that SF causes sustained induction of B(a)P biotransformation enzymes and extensive metabolism of this toxicant. As a consequence, B(a)P metabolites were generated to a greater extent in colon and liver, whose concentrations also registered a dose-dependent increase. These metabolites were found to bind with DNA and form B(a)P-DNA adducts, which may have contributed to colon tumors in a subchronic exposure regimen.

Characterization of Serum Cytokine Profile in Predominantly Colonic Inflammatory Bowel Disease to Delineate Ulcerative and Crohn's Colitides.

Background As accessible diagnostic approaches fail to differentiate between ulcerative colitis (UC) and Crohns colitis (CC) in one-third of patients with predominantly colonic inflammatory bowel disease (IBD), leading to inappropriate therapy, we aim to investigate the serum cytokine levels in these patients in search of molecular biometric markers delineating UC from CC. Methods We measured 38 cytokines, chemokines, and growth factors using magnetic-bead-based multiplex immunoassay in 25 UC patients, 28 CC patients, and 30 controls. Our results are compared with those from a review of current literature regarding advances in serum cytokine profiles and associated challenges preventing their use for diagnostic/prognostic purposes. Results Univariate analysis showed statistically significant increases of eotaxin, GRO, and TNF-α in UC patients compared to controls (Ctrl); interferon γ, interleukin (IL)-6, and IL-7 in CC group compared to Ctrl; and IL-8 in both UC and CC versus Ctrl. No cytokines were found to be different between UC and CC. A generalized linear model identified combinations of cytokines, allowing the identification of UC and CC patients, with area under the curve (AUC) = 0.936, as determined with receiver operating characteristic (ROC) analysis. Conclusions The current knowledge available about circulating cytokines in IBD is often contradictory. The development of an evidence-based tool using cytokines for diagnostic accuracy is still preliminary.

Polycyclic aromatic hydrocarbons

Livestock constitute one of the major groups of terrestrial repositories for polycyclic aromatic hydrocarbons (PAHs). From a nutrition toxicity standpoint, much emphasis had been placed on the safety of edible products derived from these animals until recently. Studies in goats, cows, and pigs have shown that PAHs undergo extensive biotransformation, and the transfer of PAH metabolites to the young depends on the lipophilicity of these compounds. The consequences of pre- and postnatal exposure to PAHs on the health of developing farm animals have not been investigated. The literature is replete with PAH toxicity in rodent models, but similar information is scarce for farm animals. Although rodents and livestock differ in their ability to process these toxicants and the manifestation of toxicity notwithstanding, the reproductive health of livestock is a major concern because PAHs are endocrine disruptors. In this chapter, an attempt has been made to critically assess the available data and put the findings into perspective without making sweeping generalizations. This chapter will contribute to increasing awareness among farm animal handlers about the harmful effects of PAHs and also aid regulatory agencies in developing appropriate risk management strategies.

Mode of action of the retrogene product SNAI1P, a SNAIL homolog, in human breast cancer cells

SNAI1P, a protein coded by a retrogene, is a member of the SNAI family of E2-box binding transcriptional repressors. To evaluate whether the mode of action of SNAI1P is similar to those of the other predominant members of the SNAI family, we studied its action on human claudin 7 (CLDN7) gene promoter which has seven E2-boxes. We over-expressed FLAG-tagged SNAI1P in MCF7 and MDA-MB-468 cells. SNAI1P inhibited the expression of CLDN7 in these recombinant cells. SNAI1P also inhibited cloned CLDN7 gene promoter activity in human breast cancer cells. ChIP assays revealed that SNAI1P is recruited on the CLDN7 gene promoter along with the co-repressor CtBP1 and the effector HDAC1. Treatment of the cells with trichostatin A, an inhibitor of HDAC1, abrogated the repressor activity of SNAI1P. These data suggest that SNAI1P inhibits CLDN7 gene promoter epigenetically in breast cancer cells through chromatin remodeling.

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.

Comparative Evaluation of Different Cell Lysis and Extraction Methods for Studying Benzo(a)pyrene Metabolism in HT-29 Colon Cancer Cell Cultures

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies.

Benzo(a)pyrene modulates fluoranthene-induced cellular responses in HT-29 colon cells in a dual exposure system.

Our environment is contaminated with a diverse array of chemicals; one of which is polycyclic aromatic hydrocarbons (PAHs). While some PAHs are potent by nature, others undergo interactions such as additivity, synergism, antagonism or potentiation to manifest their toxicity. Therefore, the objective of this study was to investigate whether exposure to benzo(a)pyrene (BaP), a PAH compound influences the cytotoxicity and metabolism of fluoranthene (FLA; another PAH compound) using HT-29 cells. Cells cultured in Dulbeccos Modified Eagle Medium were treated with 1, 5, 10, 25μM BaP and FLA (0.01% dimethylsulfoxide as vehicle) individually and in combination over the course of 0-96h. At the end of exposure, cells were stained with propidium iodide and the changes in cell cycle were analyzed using FACS analysis. Apoptosis was determined by caspase-3 assay. Post-incubation, samples were extracted and analyzed for FLA metabolites by reverse-phase HPLC with fluorescence detection. Cells exposed to BaP+FLA showed a marginal decrease in growth as compared to FLA alone and vehicle controls. Also, a decline in the percentage of cells in the S and G2 phases compared to G1 phase of cell cycle was noted when cells were treated with BaP and FLA together, compared to individual FLA treatment. The rate of FLA metabolism was more when cells were exposed to FLA in combination with BaP, compared to FLA alone. The enhanced biotransformation of FLA as a result of concomitant exposure to BaP may have implications for colon cancer risks arising from human dietary exposure to PAH mixtures through consumption of barbecued meat.

Abstract 1334: Benzo(a)pyrene concentration-dependent biotransformation and cytotoxicity in HT-29 colon cells

The majority of sporadic cancer deaths are attributed to environmental factors of which exposure to environmental toxicants is one. Benzo(a)pyrene (BaP), a member of the Polycyclic Aromatic Hydrocarbons (PAH) family of compounds is one such ubiquitous environmental toxicant; found in charcoal broiled/smoked meats, cigarette smoke, automobile exhaust, and industrial emissions. High levels of BaP have been detected in fat-rich foods such as dairy products, barbecued pork sausage, and beef meat. Since diet makes up a substantial amount of BaP intake (∼ 3 – 17 mg/person/day); ingested BaP contributing to gastrointestinal (GI) carcinogenesis deserves attention. Studies conducted using a mouse model in our laboratory has already shown that BaP contributes to colon cancer development. Through metabolic activation, BaP is biotransformed into an array of metabolites. Biotransformation of BaP proceeds through specific biochemical pathways (epoxide and quinone) each one producing specific reactive metabolites that bind with DNA, cause DNA damage and associated changes in cell cycle. The objective of this study was to determine the concentrations of BaP that are most toxic to HT-29 human colon cells and examine if any relationship exist between cellular toxicity and BaP biotransformation. Cells were cultured in Dulbecco9s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% FBS and antibiotics (penicillin/streptomycin). After revival of cells and growing them to confluence, cells were synchronized overnight by serum starvation method. The HT-29 cells were exposed to 1, 5, 10, 25, 50, and 100μM BaP over the course of 10 days and then counted using a Coulter Counter. Based on a concentration – response study 5, 10, and 25 μM BaP in DMSO (vehicle for BaP; 0.01%) were selected as optimal concentrations to analyze the effect of BaP on cell growth, viability, biochemical, and molecular endpoints 4 days after BaP exposure. The expression of both Phase I and II drug metabolizing enzymes were analyzed by western blot and RT-PCR. Analysis of cell cycle via FACS showed inhibition of the S and G2-phase in cells treated with increasing concentrations of BaP. An increase in CYP 1A1 and 1B1 expression also occurred in a BaP concentration-dependent manner. However, not much difference was seen in Phase II metabolizing enzyme expression levels regardless of the BaP concentrations tested. Our results suggest that BaP cytotoxicity is mediated by metabolic activation (funded by the NIH grants 5T32HL007735-12, 1RO1CA142845-01A1, 1S11ES014156-01A1 and Southern Regional Education Board). 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 1334. doi:10.1158/1538-7445.AM2011-1334