Identification and Comparison of Aberrant Key Regulatory Networks in Breast, Colon, Liver, Lung, and Stomach Cancers through Methylome Database Analysis

<div><p>Aberrant methylation of specific CpG sites at the promoter is widely responsible for genesis and development of various cancer types. Even though the microarray-based methylome analyzing techniques have contributed to the elucidation of the methylation change at the genome-wide level, the identification of key methylation markers or top regulatory networks appearing common in highly incident cancers through comparison analysis is still limited. In this study, we in silico performed the genome-wide methylation analysis on each 10 sets of normal and cancer pairs of five tissues: breast, colon, liver, lung, and stomach. The methylation array covers 27,578 CpG sites, corresponding to 14,495 genes, and significantly hypermethylated or hypomethylated genes in the cancer were collected (FDR adjusted p-value <0.05; methylation difference >0.3). Analysis of the dataset confirmed the methylation of previously known methylation markers and further identified novel methylation markers, such as GPX2, CLDN15, and KL. Cluster analysis using the methylome dataset resulted in a diagram with a bipartite mode distinguishing cancer cells from normal cells regardless of tissue types. The analysis further revealed that breast cancer was closest with lung cancer, whereas it was farthest from colon cancer. Pathway analysis identified that either the “cancer” related network or the “cancer” related bio-function appeared as the highest confidence in all the five cancers, whereas each cancer type represents its tissue-specific gene sets. Our results contribute toward understanding the essential abnormal epigenetic pathways involved in carcinogenesis. Further, the novel methylation markers could be applied to establish markers for cancer prognosis.</p></div

Preparation of poly(γ-glutamic acid)/hydroxyapatite monolith via biomineralization for bone tissue engineering

<div><p>A hybrid monolith of poly(γ-glutamic acid) and hydroxyapatite (PGA/HAp monolith) was prepared via biomineralization and used as a macroporous cell scaffold in bone tissue engineering. The PGA monolith having a bimodal pore size distribution was used as a substrate to induce biomineralization. The PGA/HAp monolith was obtained by immersing the PGA monolith in simulated body fluid. Pretreatment with CaCl₂ enhanced the apatite-forming ability of the PGA monolith. Murine osteoblastic MC3T3-E1 cells efficiently attached and proliferated on the PGA/HAp monolith. MTT assay showed that both the PGA and PGA/HAp monolith did not have apparent cytotoxicity. Moreover, the PGA and PGA/HAp monoliths adsorbed bone morphogenetic protein-2 (BMP-2) by electrostatic interaction which was slowly released in the medium during cell culture. The PGA/HAp monolith enhanced BMP-2 induced alkaline phosphatase activity compared to the PGA monolith and a polystyrene culture plate. Thus, these PGA/HAp monoliths may have potential in bone tissue engineering.</p></div

Cluster and heat map analysis with genes of altered methylation for five cancers.

<p>A. Cluster analysis was carried out using a methylation index (β-value) taken from Illumina Human Methylation 27 Bead Chip database registered at the GEO. Hierarchical clustering with the correlation distance was shown for 50 tumor tissues from the breast, colon, liver, lung, and stomach cancer. B. Hierarchical cluster analysis was conducted for 90 genes that were related with “cancer” from the five cancer types. The bottom panel is a heat map analysis wherein the rows and columns represent genes and cancer cell lines, respectively.</p

RT-PCR analysis of selected genes that showed altered methylation in the five cancer types.

<p>The expression level of selected genes was examined using real-time RT-PCR in a normal and a cancer cell line for the five cancer types. In case of stomach, HEK293 was used as the normal cell line. There appeared a coincidence that hypermethylated genes showed downregulation, or that hypomethylated genes showed upregulation in cancer. Each sample was examined in triplicate, and the average expression levels are presented with standard errors.</p

Highest confidence network of genes displaying altered methylation levels in breast cancer.

<p>In the network, hypermethylated genes in cancer are colored in red, whereas the hypomethylated genes are shown in green. The intensity of the color reflects the magnitude of methylation change. According to IPA, the network is relevant to “cellular movement, cellular development, and cancer.” The highest confidence network for the other four cancers is added in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097818#pone.0097818.s002" target="_blank">Fig. S2</a>. Each interaction is supported by at least one literature reference, with solid lines representing direct interactions, and dashed lines representing indirect interactions.</p

In silico expression profiles of selected genes with altered methylation in breast cancer.

<p>Five genes that have shown methylation change in breast cancer were selected, and their expression was examined in silico in the expression dataset and represented as a box plot. There appeared a coincidence that hypermethylated genes showed downregulation, or that hypomethylated genes showed upregulation in cancer. Data for the other four cancers is added in Fig. S4.</p

Differential Epigenetic Effects of Atmospheric Cold Plasma on MCF-7 and MDA-MB-231 Breast Cancer Cells

<div><p>Cold atmospheric plasma (plasma) has emerged as a novel tool for a cancer treatment option, having been successfully applied to a few types of cancer cells, as well as tissues. However, to date, no studies have been performed to examine the effect of plasma on epigenetic alterations, including CpG methylation. In this study, the effects of plasma on DNA methylation changes in breast cancer cells were examined by treating cultured MCF-7 and MDA-MB-231 cells, representing estrogen-positive and estrogen-negative cancer cells, respectively, with plasma. A pyrosequencing analysis of <i>Alu</i> indicated that a specific CpG site was induced to be hypomethylated from 23.4 to 20.3% (p < 0.05) by plasma treatment in the estrogen-negative MDA-MB-231 cells only. A genome-wide methylation analysis identified “cellular movement, connective tissue development and function, tissue development” and “cell-to-cell signaling and interaction, cell death and survival, cellular development” as the top networks. Of the two cell types, the MDA-MB-231 cells underwent a higher rate of apoptosis and a decreased proliferation rate upon plasma treatment. Taken together, these results indicate that plasma induces epigenetic and cellular changes in a cell type-specific manner, suggesting that a careful screening of target cells and tissues is necessary for the potential application of plasma as a cancer treatment option.</p></div

RT-PCR analysis of selected genes showing altered methylation after plasma treatment in breast cancer cells.

<p>Real-time RT-PCR analysis of selected genes displaying altered methylation levels in response to plasma in the MCF-7 (A) and MDA-MB-231 cells (B). Cells were treated 10 times, 30 sec each time with an hour interval between exposures, and then harvested after 24 hr. Each sample was analyzed in triplicate, with the average relative expression levels indicated with standard errors.</p

The highest confidence network of genes displaying altered methylation levels induced by plasma in the breast cancer cells.

<p>According to IPA, the highest confidence networks in the MCF-7 cells (A) and MDA-MB-231 cells (B) were “cellular movement, connective tissue development and function, tissue development” and “cell-to-cell signaling and interaction, cell death and survival, cellular development”, respectively. Genes that were hypermethylated in the plasma-treated cells are shaded in red, while those that were hypomethylated are shaded in green, with intensity signifying the magnitude of methylation change. Each interaction is supported by at least one literature reference, with solid lines representing direct interactions and dashed lines representing indirect interactions.</p

Effect of plasma on the global methylation levels in breast cell lines.

<p>The methylation levels of the four CpGs on the <i>Alu</i> from the MCF-12A, MCF-10A, MCF-7, and MDA-MB-231 cells were determined by pyrosequencing after treatment with plasma. (A) The sequence of the <i>Alu</i> adopted in this study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129931#pone.0129931.ref042" target="_blank">42</a>]. The three CpG sites analyzed are indicated in red and numbered. (B) Bar graphs showing the methylation levels of CpG #2 of <i>Alu</i> in the four cell lines. Five independent experiments were performed for each cell line and average values are given with the standard errors. (C) A representative pyrosequencing diagram for MDA-MB-231 cells.</p