College of Agriculture & Natural Resources

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    NPC1L1 knockout protects against colitis-associated tumorigenesis in mice
    (Springer Nature, 2015-03-27) He, Jianming; Shin, Hyunsu; Wei, Xing; Kadegowda, Anil Kumar G; Chen, Rui; Xie, Sandy Krystal
    Colorectal cancer is strongly associated with lipid metabolism. NPC1L1, a sterol transporter, plays a key role in modulating lipid homeostasis in vivo. Its inhibitor, ezetimibe, began to be used clinically to lower cholesterol and this caused the great debate on its role in causing carcinogenesis. Here we explored the role of NPC1L1 in colorectal tumorigenesis. Wild-type mice and NPC1L1−/− (NPC1L1 knockout) mice were treated with azoxymethane (AOM)-dextran sodium sulfate (DSS) to induce colitis-associated colorectal tumorigenesis. Mice were sacrificed 10, 15, 18 or 20 weeks after AOM treatment, respectively. Colorectal tumors were counted and analyzed. Plasma lipid concentrations were measured using enzymatic reagent kit. Protein expression level was assayed by western blot. NPC1L1−/− mice significantly had fewer tumors than wild-type. The ratio of malignant/tumor in NPC1L1−/− mice was significantly lower than in wild-type 20 weeks after AOM-DSS treatment. NPC1L1 was highly expressed in the small intestine of wild-type mice but its expression was undetectable in colorectal mucous membranes or tumors in either group. NPC1L1 knockout decreased plasma total cholesterol and phospholipid. NPC1L1−/− mice had significant lower intestinal inflammation scores and expressed inflammatory markers p-c-Jun, p-ERK and Caspase-1 p20 lower than wild-type. NPC1L1 knockout also reduced lymphadenectasis what may be caused by inflammation. NPC1L1 knockout in mice decreased β-catenin in tumors and regulated TGF-β and p-gp in adjacent colons or tumors. There was not detectable change of p53 by NPC1L1 knockout. Our results provide the first evidence that NPC1L1 knockout protects against colitis-associated tumorigenesis. NPC1L1 knockout decreasing plasma lipid, especially cholesterol, to reduce inflammation and decreasing β-catenin, p-c-Jun and p-ERK may be involved in the mechanism.
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    Selenium compounds activate early barriers of tumorigenesis
    (2011) WU, MIN; Cheng, Wen-Hsing; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Selenium chemoprevention by apoptosis has been well studied, but it is not clear whether selenium can activate early barriers of tumorigenesis, namely senescence and DNA damage response. To test this hypothesis, we treated normal and cancerous cells with a gradient concentration of sodium selenite, methylseleninic acid and methylselenocysteine for 48 h, followed by a recovery of 1-7 days. Here we show that selenium compounds at doses of ≤LD50 can induce cellular senescence, as evidenced by the expression of senescence-associated β-galactosidase and 5-bromo-2-deoxyuridine incorporation, in normal but not cancerous cells. In response to clastogens, the ataxia telangiectasia mutated (ATM) protein is rapidly activated, which in turn initiates a cascade of DNA damage response. We found that the ATM pathway is activated by the selenium compounds, and the kinase activity is required for the selenium induced senescence response. Pretreatment of the MRC-5 non-cancerous cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl suppresses the selenium induced ATM activation and senescence. Taken together, the results suggest a novel role of selenium in the activation of early tumorigenesis barriers specific in non-cancerous cells, whereby selenium induces an ATM-dependent senescence response that depends on reactive oxygen species. The tumor suppressor p53 is a substrate of the ATM kinase and plays an important role in senescence. To determine mechanism by which selenium induces the ATM-dependent senescence, we employed shRNA knockdown approach and other DNA damage assays to determine the role of p53 in the senescence response. Results from senescence-associated expression of β-galactosidase assay indicate that p53 shRNA MRC-5 cells did not show senescent phenotype with a series of concentrations of methylseleninic acid (0-10 μM) after 7-day recovery. However, loss of p53 renders MRC-5 cells more resistant to MSeA treatment and increased its genomic instability. We also observe that MSeA can cause increased irreversible G2/M arrest in scramble MRC-5 cells but treated p53 shRNA MRC-5 can recover back to non-treated status after 7-day recovery. Taken together, p53 is involved in the ATM-dependent senescence in the response of MRC-5 normal cells to selenium compounds.
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    Modulation of Human Tumor Suppressor Genes, Gadd45, p53 and p38 MAPK by Zinc Status in Normal Human Bronchial Epithelial Cells
    (2007-05-08) Shih, Sheung-Mei; Lei, David K.Y.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The effect of zinc status on the cell signaling transduction of tumor suppressor genes, Growth Arrest and DNA Damage inducible gene (Gadd45), p53, and p38 Mitogen Activated Protein Kinase (MAPK) were examined in Normal Human Bronchial Epithelial (NHBE) cells. Cells were cultured for one passage in different concentrations of zinc: < 0.4 μM (ZD, zinc-deficient); 4 μM (ZN, zinc normal) as normal zinc level found in most culture medium; 16 μM (ZA, zinc adequate) represented normal human plasma zinc level; and 32 μM (ZS, zinc supplementation) represented the optimal plasma zinc attainable by oral supplementation. Cell growth inhibition, up-regulation of Gadd45, p53 and p38 MAPK mRNA and protein expressions, and blockage of G2/M cell cycle progression were observed in ZS cells. The siRNA-mediated knocking down of Gadd45 was found to alleviate G2/M blockage partially in ZS cells, which indicated that the blockage is partially Gadd45 dependent. In ZS cells, the enhanced phosphorylation of p38 MAPK and p53 were abrogated after suppressing Gadd45 by siRNA, implicating that the enhanced phosphorylation of p53 and p38 MAPK were Gadd45 dependent. By using p53 transactivation inhibitor Pifithrin, the upregulated Gadd45 protein, the enhanced Gadd45 promoter activity, and the reduced level of CDK1/Cyclin B1 complex were all restored back to normal levels in ZS cell, implying that these ZS induced changes were p53 dependent. Furthermore, the ZS induced upregulation of Gadd45 expression, displacement of CDC25B from nucleus to cytoplasm, reduction of CDK1/Cyclin B1 complex level, enhancement of the activation and phosphorylation of p53, and delay of G2/M cell cycle progression were normalized by p38 MAPK dominant negative and protein inhibitor SB202190. Thus, the ZS induced changes were dependent on the activation of p38 MAPK. Our data support the involvement of a positive Gadd45, p53 and p38 MAPK feedback loop in response to stress induced by zinc supplementation. These findings demonstrate the importance of p38 MAPK and p53 in the regulation of G2/M cell cycle progression in response to the stress induced by high zinc via Gadd45 and cell cycle checkpoint regulatory proteins, including CDK1, Cyclin B1 and CDC25B.