Proteins in Colonic Epithelial Cells

Consuming specific dietary substances, like probiotic bacteria and flavonoids, may enhance intestinal health. These projects examine the potential mechanism by which probiotic bacteria and flavonoids modulate cell signaling events associated with inflammation and cell migration, respectively. By causing these events, these dietary factors could decrease the number of preneoplastic cells at risk for transformation into tumor cells. These data offer a new potential mechanism by which dietary factors may decrease risk of colorectal cancer.

Project A: To determine the effects of probiotic lactic acid bacteria on pro-inflammatory mediator production in YAMC and IMCE cells, we will examine the ability of irradiated strains of Lactobacillus spp. to modulate cytokine production in mouse colon epithelial cell lines YAMC and IMCE. The effects of these bacteria on the proinflammatory cytokines nitric oxide (NO), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-alpha (TNF-alpha) will be measured. The signaling pathways involved in the inhibitory response to probiotic bacterial exposure will be explored with the use of enzymatic inhibitors to relevant targets. Projects B and C: 1. To determine if dietary flavonoids will demonstrate structure-activity relationships with regard to the induction of the migratory phenotype in YAMC and IMCE cells. We will test this hypothesis by performing dose-response studies of flavonoid-dependent two-dimensional cell migration in YAMC and IMCE cells using twelve (12) compounds representing five (5) flavonoid families. 2. To determine flavonoids which induce the migratory phenotype do so by activating specific intracellular signaling pathways that originate from their binding to cellular receptors. We will test this hypothesis by co-treating YAMC and IMCE cells with migration-inducing flavonoids and receptor- and signaling pathway-specific antagonists or inhibitors. The time- and dose-dependence of the inhibitory effects of these antagonists/inhibitors on flavonoid-induced migration will yield critical evidence about the aggregate effects of different signaling mechanisms on cell migration. 3. To determine if flavonoids which induce the migratory phenotype will modulate the expression of genes related to cell differentiation. Total RNA samples from YAMC and IMCE cells treated over a 48 hours time course with four migration-inducing flavonoids will be used to produce cDNA probes for microarray analysis using the National Institute of Aging 15K mouse set. These experiments will identify genes and clusters of gene families whose transcription is increased or decreased in response to these flavonoids. Genes or gene clusters recruited or inhibited in response to exposure to dietary factors will be correlated with migratory responses to identify functional genomic determinants of cell differentiation. 4. To determine, by gene knockdown techniques (e.g., small interfering RNA analysis), if specific cell signaling pathways contribute additively or synergistically to flavonoid-induced gene expression associated with cell migration.

This progress report details effort on two projects involving the potential of two types of dietary constituents to modulate biological processes related to colon cancer prevention in a cell culture model system. The first project has established, for the first time, the role of the spice component, curcumin, in causing cell migration in colonic epithelial cells. Colonic epithelial cell migration is required for movement up the crypt-villus axis and hence, normal differentiated cell function. This migratory phenotype is dependent upon wild-type adenomatous polyposis coli (Apc) expression. Non-tumorigenic murine colon epithelial cell lines with distinct Apc genotypes, YAMC (Apc+/+) and IMCE (ApcMin/+) cells) were used to assess the association between Apc genotype, cell motility and matrix metalloproteinase (MMP) activity in these cells. YAMC and IMCE cells were treated with epidermal growth factor (EGF; 1, 10 and 25 ng/ml) or hepatocyte growth factor (HGF; 1, 10 and 25 ng/ml) and/or curcumin (0.1-100 microM). EGF (25 ng/ml) and HGF (25 ng/ml) induced a greater migratory response in YAMC compared to IMCE cells after 24 hours (p<0.05). Treatment with curcumin induced an equivalent or greater migratory response in IMCE than YAMC cells. When migrating cells were treated with an inhibitor of matrix metalloproteinases (Ilomastatr?; I), migration was inhibited in both cell types. Curcumin-induced migration was inhibited by I in both cell types at the highest dose (50 microM), while the lower doses (10 and 25 microM) had minimal effects. These data provide the first evidence that colon epithelial cells heterozygous for Apc are less migratory than cells containing wild-type Apc. This migration is dependent on MMP activity and likely due specifically to changes in membrane type-1-MMP. The second project in which significant progress has been made utilizes the same cell culture models described above. These studies have identified specific lactic acid bacteria (LAB) which can decrease the production of proinflammatory mediators caused by exposure of these cells to bacterial pathogen, Salmonella dublin. Probiotic bacteria may inhibit inflammatory processes resulting from exposure to bacterial pathogens by blocking nuclear factor kappa B (NF-kB) nuclear translocation and nitric oxide (NO) production. The objective of this study was to assess the ability of two probiotic bacteria, Lactobacillus casei (LC) and Lactobacillus reuteri (LR), to block Salmonella dublin-induced NF-kB translocation and NO production in a non-tumorogenic murine colon epithelial cell line. Nuclear translocation of NF-kB, was assessed by immunocytochemistry and western blotting; NO was assessed in culture supernatants using the Greiss reaction. LC and LR differentially inhibited nuclear translocation of NF-kB and NO production induced by S. dublin. Western blots from nuclear extracts confirmed that LR was more effective than LC in blocking S. dublin-induced NF-kB translocation.