Association of aberrant DNA methylation in Apc(min/+) mice with the epithelial-mesenchymal transition and Wnt/ß-catenin pathways: genome-wide analysis using MeDIP-seq.

BACKGROUND: Aberrant DNA methylation at the 5-carbon on cytosine residues (5mC) in CpG dinucleotides is probably the most extensively characterized epigenetic modification in colon cancer. It has been suggested that the loss of adenomatous polyposis coli (APC) function initiates tumorigenesis and that additional genetic and epigenetic events are involved in colon cancer progression. We aimed to study the genome-wide DNA methylation profiles of intestinal tumorigenesis in Apc(min/+) mice. RESULTS: Methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing was used to determine the global profile of DNA methylation changes in Apc(min/+) mice. DNA was extracted from adenomatous polyps from Apc(min/+) mice and from normal intestinal tissue from age-matched Apc(+/+) littermates, and the MeDIP-seq assay was performed. Ingenuity Pathway Analysis (IPA) software was used to analyze the data for gene interactions. A total of 17,265 differentially methylated regions (DMRs) displayed a ≥ 2-fold change (log2) in methylation in Apc(min/+) mice; among these DMRs, 9,078 (52.6 %) and 8,187 (47.4 %) exhibited increased and decreased methylation, respectively. Genes with altered methylation patterns were mainly mapped to networks and biological functions associated with cancer and gastrointestinal diseases. Among these networks, several canonical pathways, such as the epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin pathways, were significantly associated with genome-wide methylation changes in polyps from Apc(min/+) mice. The identification of certain differentially methylated molecules in the EMT and Wnt/ß-catenin pathways, such as APC2 (adenomatosis polyposis coli 2), SFRP2 (secreted frizzled-related protein 2), and DKK3 (dickkopf-related protein 3), was consistent with previous publications. CONCLUSIONS: Our findings indicated that Apc(min/+) mice exhibited extensive aberrant DNA methylation that affected certain signaling pathways, such as the EMT and Wnt/ß-catenin pathways. The genome-wide DNA methylation profile of Apc(min/+) mice is informative for future studies investigating epigenetic gene regulation in colon tumorigenesis and the prevention of colon cancer.

In vitro and in vivo anti-inflammatory effects of a novel 4,6-bis ((E)-4-hydroxy-3-methoxystyryl)-1-phenethylpyrimidine-2(1H)-thione.

Inflammation plays a critical defensive role in the human body. However, uncontrolled or aberrant inflammatory responses contribute to various acute and chronic diseases. The Nrf2-ARE pathway plays a pivotal role in the regulation of inflammatory markers, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). On the basis of this concept, we synthesized a novel anti-inflammatory 4,6-bis ((E)-4-hydroxy-3-methoxystyryl)-1-phenethylpyrimidine-2(1H)-thione (HPT), and in vitro experiments using HepG2-C8 ARE-luciferase-transfected cells demonstrated the induction of Nrf2-ARE activity. In lipopolysaccharide (LPS)-induced RAW 264.7 cells, HPT treatment reduced the production of nitric oxide (NO) as well as the protein and mRNA expression levels of COX-2 and iNOS, in a dose-dependent manner. In addition, HPT suppressed the mRNA expression of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6. In LPS-induced macrophages, HPT inhibited COX-2 and iNOS by blocking the activation of p38 and c-Jun NH2-terminal kinase (JNK) but not extracellular signal-regulated kinase (ERK1/2). Furthermore, an in vivo anti-inflammatory study was performed using a TPA-induced skin inflammation mouse model, and the results showed that HPT reduced TPA-induced inflammation and attenuated the expression of COX-2 and iNOS in TPA-induced mouse skin tissue. Thus, HPT demonstrated anti-inflammatory activity both in LPS-induced RAW 264.7 cells and TPA-stimulated mouse skin and may therefore serve as a potential anti-inflammatory agent.

Pharmacodynamics of ginsenosides: antioxidant activities, activation of Nrf2, and potential synergistic effects of combinations.

Ginseng has long been used in Asian countries for more than 2000 years. Currently, in the "Western World or Western Medicines", many reports have indicated that they have used herbal medicines, and ginseng is one of the most popular herbs. Several recent reports have indicated that the antioxidant/antioxidative stress activities of ginseng play a role in the benefits of ginseng; however, the precise mechanism is lacking. The antioxidant response element (ARE) is a critical regulatory element for the expression of many antioxidant enzymes and phase II/III drug metabolizing/transporter genes, mediated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The aim of this study was to examine the potential activation and synergism of Nrf2-ARE-mediated transcriptional activity between three common ginsenosides present in ginseng, ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), and ginsenoside 20(S)-protopanaxatriol (20S). We tested whether these ginsenosides and their combinations could induce Nrf2-ARE activities in HepG2-C8 cells with stably transfected ARE luciferase reporter gene. Cell proliferation, antioxidant and ARE activities, Western blotting of Nrf2 protein, and qPCR of mRNA of Nrf2 were conducted for Rb1, Rg1, and 20S as well as the combinations of 20S with Rb1 or Rg1. To determine the combination effects, the combination index (CI) was calculated. Rb1 and Rg1 are relatively nontoxic to the cells, while 20S at 50 µM or above significantly inhibited the cell proliferation. Rb1, Rg1, or 20S induced total antioxidant activity and ARE activity in a concentration-dependent manner. Furthermore, combinations of 20S with either Rb1 or Rg1 induced total antioxidant and ARE activity synergistically. The induction of Nrf2 protein and mRNA was also found to be synergistic with the combination treatments. In summary, in this study, we show that ginsenosides Rb1, Rg1, and 20S possess antioxidant activity, transcriptionally activating ARE as well as the potential of synergistic activities. The Nrf2-ARE-mediated antioxidant pathway could play a role for the overall antioxidative stress activities, which could be important for ginseng's health beneficial effects such as cancer chemopreventive activities.