Peracetylated (−)-Epigallocatechin-3-gallate (AcEGCG) Potently Suppresses Dextran Sulfate Sodium-Induced Colitis and Colon Tumorigenesis in Mice

Previous studies reported that peracetylated (-)-epigallocatechin-3-gallate (AcEGCG) has antiproliferative and anti-inflammatory activities. Here, we evaluated the chemopreventive effects and underlying molecular mechanisms of dietary administration of AcEGCG and EGCG in dextran sulfate sodium (DSS)-induced colitis in mice. The mice were fed a diet supplemented with either AcEGCG or EGCG prior to DSS induction. Our results indicated that AcEGCG administration was more effective than EGCG in preventing the shortening of colon length and the formation of aberrant crypt foci (ACF) and lymphoid nodules (LN) in mouse colon stimulated by DSS. Our study observes that AcEGCG treatment inhibited histone 3 lysine 9 (H3K9) acetylation but did not affect histone acetyltransferase (HAT) activity and acetyl- CREB-binding protein (CBP)/p300 levels. In addition, pretreatment with AcEGCG decreased the proinflammatory mediator levels by down-regulating of PI3K/Akt/NFκB phosphorylation and p65 acetylation. We also found that treatment with AcEGCG increased heme oxygenase-1­(HO-1) expression via activation of extracellular signal-regulated protein kinase (ERK)­1/2 signaling and acetylation of NF-E2-related factor 2 (Nrf2), thereby abating DSS-induced colitis. Moreover, dietary feeding with AcEGCG markedly reduced colitis-driven colon cancer in mice. Taken together, these results demonstrated for the first time the in vivo chemopreventive efficacy and molecular mechanisms of dietary AcEGCG against inflammatory bowel disease (IBD) and potentially colon cancer associated with colitis. These findings provide insight into the biological actions of AcEGCG and might establish a molecular basis for the development of new cancer chemopreventive agents

Inhibitory effects of single predicted drugs on colon cancer and breast cancer cell lines.

<p>–: not detected from 0.1 to 30 µM.</p

Function-function networks for colorectal adenoma.

<p>Condition specific function-function networks (FFNs) were generated from gene-gene networks (GGINs), shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086299#pone.0086299.s002" target="_blank">Figure S2</a>, by reduction. Nodes in an FFN are functional modules (FMs), which are gene sets in the corresponding GGIN forming over-represented Gene Ontology terms. FMs containing less than 70 genes are not shown. The diameter of a node scales with the logarithm of the number of genes in the node. The color shade of a node indicates the number of intra-node gene-gene interactions per gene. The thickness of the edge indicates the number of inter-node gene-gene interactions.</p

miR-200c and GATA binding protein 4 regulate human embryonic stem cell renewal and differentiation

Human embryonic stem cells (hESCs) are functionally unique for their self-renewal ability and pluripotency, but the molecular mechanisms giving rise to these properties are not fully understood. hESCs can differentiate into embryoid bodies (EBs) containing ectoderm, mesoderm, and endoderm. In the miR-200 family, miR-200c was especially enriched in undifferentiated hESCs and significantly downregulated in EBs. The knockdown of the miR-200c in hESCs downregulated Nanog expression, upregulated GATA binding protein 4 (GATA4) expression, and induced hESC apoptosis. The knockdown of GATA4 rescued hESC apoptosis induced by downregulation of miR-200c. miR-200c directly targeted the 3′-untranslated region of GATA4. Interestingly, the downregulation of GATA4 significantly inhibited EB formation in hESCs. Overexpression of miR-200c inhibited EB formation and repressed the expression of ectoderm, endoderm, and mesoderm markers, which could partially be rescued by ectopic expression of GATA4. Fibroblast growth factor (FGF) and activin A/nodal can sustain hESC renewal in the absence of feeder layer. Inhibition of transforming growth factor-β (TGF-β)/activin A/nodal signaling by SB431542 treatment downregulated the expression of miR-200c. Overexpression of miR-200c partially rescued the expression of Nanog/phospho-Smad2 that was downregulated by SB431542 treatment. Our observations have uncovered novel functions of miR-200c and GATA4 in regulating hESC renewal and differentiation

Enrichment score versus fold-change for CMap drugs.

<p>Enrichment score (ES) was obtained by querying the CMap with gene set (size indicated by vertical bar) determined using varying fold-change (FC) threshold. A drug is considered beneficial for the treatment for colorectal adenoma if ES <−0.5, harmful if ES >0.3, and neutral otherwise. (A) Screening by IGCM procedure. Querying gene set was complete set of differentially expressed genes (DEGs) identified from gene expression arrays of colorectal adenoma cohort (versus control) using the SAM algorithm with fixed FDR <0.01. (B) Screening by FMCM procedure. Querying gene sets were functional modules obtained by partition of over-represented Gene Ontology terms in GSToP filtered DEGs.</p

Clustering of genomic profiles of drug-treated cancer cell lines HCT116 and MCF7.

<p>(A) Individual gene approach (IGA). (B) Gene-set approach (GSA). Cell lines were treated with three drugs: GW-8510, phenoxybenzamine (PB), and imipenem. Entries marked “cmap” were microarray drug treatment genomic profiles of MCF7 taken from the CMap. Others were from drug treatment microarray experiments (Affymetrix U219 (PrimeView) platform) conducted for the present study, where the same experimental protocol used in CMap were followed: averaged over three dosages of 10 M, 11.8 M, 13.4 M; treatment time 6 hours after overnight culture. Heatmaps were results of two-way hierarchical clustering.</p

Overlap of candidate repurposed drug sets curated from colon adenoma and colorectal cancer data sets.

<p>Numbers in brackets correspond to those given in the first column of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086299#pone-0086299-t001" target="_blank">Table 1</a>, which lists the drug set for colon adenoma. The overlap includes 9 of the 13 drugs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086299#pone-0086299-t001" target="_blank">Table 1</a> with degrees not less than 3, and 5 of the 8 drugs selected for cell viability tests marked by “*”.</p

Function-drug association map (FDAM) for colorectal adenoma.

<p>Nodes in the map are functional modules (FMs; gene sets) and drugs obtained by querying CMap using individual FMs. Drug-function links indicate beneficial (green) or harmful (red). Only drugs beneficial to at least one FM are included.</p

Viability test of colon and breast cancer cells treated with single drug.

<p>Tests were conducted on eight drugs: phenoxybenzamine (PB), GW-8510, phthalylsulfathiazole (PS), etacrynic acid (EA), ginkgolide A (GA), triflusal (TF), imipenem (IM), and 6-azathymine (6-AT), with concentrations of 0, 0.1, 1, 10, and 30 µM. (A) Viability of MCF7 on treatment of the eight drugs. (B) Viability of five cell lines on treatment of GW-8510. Colon cancer cells HCT116, RKO, SW403 and SW620, and the breast cancer cell MCF7, were treated with single drug for 5 days. After 5 days, proliferation activities of these cells were detected by Alamar Blue assay.</p