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United States Department of Agriculture

Agricultural Research Service


Location: Diet, Genomics and Immunology Laboratory

2011 Annual Report

1a. Objectives (from AD-416)
The overall goal of the project is to elucidate the molecular and cellular mechanisms that respond to selected health promoting food components to reduce the risk of chronic diseases such as cancers and obesity. A secondary aim is to explore the utility of a porcine model to test the effect of health maintenance via diet and identify resulting biomarkers that reflect health status. Objective 1. Elucidate biological activities of health promoting phytochemicals from grape, soy, and cruciferous vegetables against development of breast and prostate cancer. Objective 2. Identify molecular targets and mechanisms of action of health promoting food components in animal or in vitro models of cancer and obesity. Objective 3. Ascertain the effects of specific probiotic strains in appropriate animal models of obesity. Objective 4. Identify plant polyphenols and probiotics that affect adipocyte numbers, size, and fat accumulation, and the regulation of proinflammatory mRNA stability by tristetraprolin. Objective 5. Tie together obesity, inflammation, and cancer mechanistically in appropriate animal or in vitro models.

1b. Approach (from AD-416)
Studies will evaluate if phytoalexins structurally similar to resveratrol exerts similar anti-prostate cancer effects; if soy phytoalexin glyceollins exert anti-prostate cancer effects; if phytochemicals modulate LXR-mediated pathways in prostate epithelial cells and modulate LXR-mediated pathways in macrophage. Other studies will determine if probiotic bacterial strains differ in their protective effects against chronic diseases related to obesity; regulate adipocyte numbers, size, and fat accumulation associated with the anti-inflammatory protein tristetraprolin (TTP); if obesity alters the macrophage phenotype and function in adipose tissue, colon, breast, and prostate following increased localized inflammation; and if broccoli-derived phytochemicals modulate LXR-responsive pathways in vivo. The studies will involve in vitro cell culture approaches confirmed by rodent and pig animal models.

3. Progress Report
We hypothesized that modulation of androgen and/or cholesterol-related pathways by cruciferous vegetable-derived compounds indole-3-carbinol (I3C) and diindolylmethane (DIM) may be potential mechanisms by which these bioactive food components exert their protective effects against prostate cancer. We focused on the effects of I3C and DIM on Liver X receptors (LXRs)-mediated pathways. LXR-alpha and beta are transcriptional factors that use cholesterol metabolites oxysterol as ligands and serve as critical regulators of cholesterol metabolism. It was found that DIM, but not I3C, induces the transcripts of two LXR-responsive ATP-binding cassette transporters involved in cholesterol efflux, ABCG1 and ABCA1 mRNA, in the androgen responsive human prostate cancer cell LNCaP. Exposure of LNCaP cells to 5 microM DIM for 48 hours lead to a 5-fold increase in ABCG1 and ABCA1 mRNA levels. Knock-down of LXR expression using siRNA against both alpha and beta isoforms abrogated the ability of DIM to induce ABCG1 but not ABCA1 mRNA, supporting the effects of DIM on ABCG1 is mediated through LXR-associated pathway. However, we did not observe induction of ABCG1 or ABCA1 mRNA in androgen non-responsive human prostate cancer cells PC3 treated with DIM. Furthermore, in the LNCaP cells, knock-down of the androgen receptor (AR) expression with AR siRNA blocked DIM induction of ABCA1 and ABCG1 mRNAs. In summary, our results suggest that DIM may play a role in modulating cholesterol metabolism, in part via androgen-mediated pathway in prostate cancer cells. However, since DIM modulation of ABCG1 was LXR-dependent, it is likely that multiple pathways are involved with DIM modulation of cholesterol metabolism. Furthermore, these effects are unique to DIM, but not I3C, suggesting these two dietary components may act through different mechanisms for their cancer preventive effects. To further the understanding of how phytochemicals may prevent cancer, procedures were expanded to detect how these nutrients effect the regulatory proteins that control growth in both normal and tumor cells. The phytochemicals tested in our model cellular systems, such as resveratrol, have been shown to be more effective in modulating the regulatory proteins in cancer cell lines rather than in normal cell lines. By observing changes in proteins such as p21, p27, hypophosphorylated Rb, and cyclins B,D, and E, these procedures can help characterize the potential for anti-cancer of phytochemicals.

4. Accomplishments
1. With the advent of high-throughput sequencing methods, there is an increased interest in the fields of metagenomics and microbiomics. Often a researcher is interested in knowing which features, such as operational taxonomic units and clusters of orthologous genes, in a microbiome or metagenome dataset are differentially abundant. It can be difficult, however, for a researcher to know which statistical method is most appropriate for a particular dataset. This study compared the suitability of several statistical methods for detecting differentially abundant features in high-throughput microbiome and metagenome datasets. Amongst the statistical methods tested, an arcsine square root data transformation followed by the t Test (one-factor case), Gaussian generalized linear model (one- and two-factor cases), or ANOVA Type II sum of squares (2-factor case) are recommended for high-throughput microbiome and metagenome data quantified as relative abundances.

2. There has been growing interest in examining the immune-modulating effects of some strains of lactic acid producing probiotic bacteria on the gut micro-biota. Data from human and animal studies have established that some commensal derived probiotic bacteria can decrease expression of pro-inflammatory mediators and lower serum cholesterol levels by decreasing entrohepatic recirculation of bile acids and cholesterol. There is little characterization of the systemic effects of probiotics on cholesterol metabolism in extra-hepatic tissue. Recent evidence suggests that probiotics can modulate cholesterol absorption through interactions with LXRa and NPC1L implicating effects on the LXR axis in cell types outside of the gut. We, therefore, examined whether alveolar macrophages (AM) isolated from lungs of juvenile Ossabaw pigs fed either a high fat (HF) or HF diet supplemented with the probiotic (PB) Lactobacillus paracasei changed cholesterol metabolism and expression of LXR and inflammatory related genes and proteins. AM isolated from pigs fed the HF diet had significantly higher concentrations of cholesterol esters (CE) compared with AM from pigs fed a control basal diet, but this effect was not observed in AM from pigs fed a HF diet given a daily treatment with PB. Ex vivo stimulation of AM with LPS significantly opposed LXR agonist-mediated transcription of cholesterol metabolism genes. This effect was abrogated for ABCA1, CH25H, and PPARy expression in AM from pigs fed PB. We observed that AM from pigs fed a HF diet had a significantly higher IL-1ß mRNA response to LPS. This was not affected by PB. Moreover, AM from pigs fed a control diet supplemented with PB had significantly higher mRNA levels of IL-6 in response to LPS. Taken together, these data demonstrated a role for probiotic L. paracasei in modulating AM cholesterol metabolism and response to inflammation.

3. Food intake and energy expenditure are regulated by the central nervous system. Specifically, the hypothalamus plays an important role in the regulation of satiety and appetite through leptin and insulin signaling. Leptin, a cytokine secreted by adipocytes, is known to reduce food intake and increase energetic expenditure. Hyperleptinemia, an excess of leptin production, and leptin resistance, a decreased response to exogenous leptin, have been observed in obesity. Although many obesity studies revealed important mechanisms of leptin resistance in hypothalamus, most of the studies have been done in small animal models such as mice and rats. Compared to the rodent brain, the pig brain more closely resembles the human brain in terms of both anatomy and biochemistry, which associates the pig with a higher translational value. We recently established an ex vivo functional assay using sections of pig brain to examine leptin response. Brain slices from pig hypothalamus were cultured with or without human recombinant leptin and measured for phosphorylation of STAT3, which is considered as an indicator of leptin signaling in vivo. A dose-dependent increase of p-STAT3 was found with increasing levels of leptin added to hypothalamus. These preliminary results demonstrated that short term ex-vivo culture of hypothalamus sections constitutes an ex-vivo functional assay to examine leptin/insulin signaling in brain from pigs fed different obesogenic diets. Currently, juvenile pigs were fed three different obesogenic diets containing excess calories from a fructose, fat, or a combination of high fat and fructose, and brain sections from this study will be used to investigate the effect of diet composition on leptin signaling.

4. Five Gynostemma pentaphyllum (GP) samples were investigated and compared for their chemical compositions and their antioxidant, antiproliferative, and anti-inflammatory effects. Extracts (50% acetone, 75% ethanol, and 100% ethanol) of the five GP samples (GP1-5) differed in their total phenolic, saponin, and flavonoid contents and in their rutin and quercetin concentrations. The highest level of total flavonoids was 63.5 mg of rutin equiv/g in GP4, and the greatest total phenolic content was 44.3 mg of gallic acid equiv/g in GP1 with 50% acetone as the extraction solvent. GP2 had the highest total saponin content of 132.6 mg/g with 100% ethanol as the extraction solvent. These extracts also differed in their scavenging capacity against DPPH and hydroxyl radicals, although they all showed significant radical scavenging capacity. The 100% ethanol extracts also showed dose dependent inhibition of IL-6 and Ptgs2 mRNA expression and weak inhibition on TNF-R mRNA expression. In addition, GP1 had the highest antiproliferative activity at 3.2 mg equiv/mL concentration in HT-29 human colon cancer cells. The results will be used to study the use of products derived from G. pentaphyllum for improving human health.

5. Engeletin and astilbin, flavonoid compounds, were isolated from the leaves of Engelhardia roxburghiana for the first time. The chemical structures of engeletin and astilbin were confirmed by 1H and 13C nuclear magneticresonance (NMR) and mass spectrometry (MS) spectra, and their anti-inflammatory activities were studied in lipopolysaccharide (LPS)-stimulated mouse J774A.1 macrophage cells. LPS induced the inflammatory state in macrophage cells and increased mRNA expressions of pro-inflammatory cytokines. Engeletin and astilbin exhibited remarkable inhibitory effects on interleukin (IL)-1ß and IL-6 mRNA expression. Significant inhibition of LPS-mediated mRNA expressions were also seen in LPS binding toll-like receptor (TLR)-4, pro-inflammatory cytokine tumor necrosis factor (TNF)-R, IL-10, chemoattractant monocyte chemotactic protein (MCP)-1, and cyclooxygenase (COX)-2 genes. The reduced expression of these cytokines may modulate the immune response and reduce inflammatory activation, indicating that engeletin and astilbin may serve as potential anti-inflammatory agents.

Review Publications
Qi, Y., Schoene, N.W., Lartey, F., Cheng, W. 2011. Selenium compounds activate ATM-dependent DNA damage responses via the mismatch repair protein hMLH1 in colorectal cancer cells. Journal of Biological Chemistry. 285(43):33010-33017.

Huang, H., Cheng, Z., Shi, H., Xin, W., Wang, T.T., Yu, L. 2011. Isolation and characterization of two flavonoids, engeletin and astilbin, from the leaves of Engelhardia roxburghiana, and their potential anti-inflammatory properties. Journal of Agricultural & Food Chemistry. 59(9):4562-9.

Wang, T.T., Schoene, N.W., Milner, J.A., Kim, Y.S. 2011. Broccoli-derived phytochemicals indole-3-carbinol and 3,3’-diindolylmethane exert concentration-dependent pleiotropic effects on prostate cancer cells: Comparison with other cancer preventive phytochemicals. Molecular Carcinogenesis. DOI: 10.1002/mc.20774.

Xie, Z., Liu, W., Huang, H., Slavin, M., Zhao, Y., Whent, M., Blackford, J., Lutterodt, H., Zhou, H., Chen, P., Wang, T.T., Liangli, Y. 2010. Chemical composition of five commercial gynostemma pentaphyllum samples and their radical scavenging, antiproliferative, and anti-inflammatory properties. Journal of Agricultural and Food Chemistry. 58(21):11243-11249.

Last Modified: 09/18/2017
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