Jun Terashima

Analysis of DNA methylation landscape reveals the roles of DNA methylation in the regulation of drug metabolizing enzymes.

BackgroundDrug metabolizing enzymes (DMEs) exhibit dramatic inter- and intra-individual variability in expression and activity. However, the mechanisms determining this variability have not been fully elucidated. The aim of this study was to evaluate the biological significance of DNA methylation in the regulation of DME genes by genome-wide integrative analysis.ResultsDNA methylation and mRNA expression profiles of human tissues and hepatoma cells were examined by microarrays. The data were combined with GEO datasets of liver tissues, and integrative analysis was performed on selected DME genes. Detailed DNA methylation statuses at individual CpG sites were evaluated by DNA methylation mapping. From analysis of 20 liver tissues, highly variable DNA methylation was observed in 37 DME genes, 7 of which showed significant inverse correlations between DNA methylation and mRNA expression. In hepatoma cells, treatment with a demethylating agent resulted in upregulation of 5 DME genes, which could be explained by DNA methylation status. Interestingly, some DMEs were suggested to act as tumor-suppressor or housekeeper based on their unique DNA methylation features. Moreover, tissue-specific and age-dependent expression of UDP-glucuronosyltransferase 1A splicing variants was associated with DNA methylation status of individual first exons.ConclusionsSome DME genes were regulated by DNA methylation, potentially resulting in inter- and intra-individual differences in drug metabolism. Analysis of DNA methylation landscape facilitated elucidation of the role of DNA methylation in the regulation of DME genes, such as mediator of inter-individual variability, guide for correct alternative splicing, and potential tumor-suppressor or housekeeper.

An aryl hydrocarbon receptor induces VEGF expression through ATF4 under glucose deprivation in HepG2.

BackgroundAryl hydrocarbon receptor (AhR) not only regulates drug-metabolizing enzyme expression but also regulates cancer malignancy. The steps to the development of malignancy include angiogenesis that is induced by tumor microenvironments, hypoxia, and nutrient deprivation. Vascular endothelial growth factor (VEGF) plays a central role in the angiogenesis of cancer cells, and it is induced by activating transcription factor 4 (ATF4).ResultsRecently, we identified that glucose deprivation induces AhR translocation into the nucleus and increases CYP1A1 and 1A2 expression in HepG2 cells. Here, we report that the AhR pathway induces VEGF expression in human hepatoblastoma HepG2 cells under glucose deprivation, which involves ATF4. ATF4 knockdown suppressed VEGF expression under glucose deprivation. Moreover, AhR knockdown suppressed VEGF and ATF4 expression under glucose deprivation at genetic and protein levels.ConclusionsThe AhR-VEGF pathway through ATF4 is a novel pathway in glucose-deprived liver cancer cells that is related to the microenvironment within a cancer tissue affecting liver cancer malignancy.

Induction of CYP1 family members under low-glucose conditions requires AhR expression and occurs through the nuclear translocation of AhR.

Cross-talk between the aryl hydrocarbon receptor (AhR) pathway and the typical stress response is thought to be an important signal transduction in response to nutrient-stress conditions, such as glucose deprivation in liver cells. In the present study, we demonstrate that reduction of glucose concentration in the medium of HepG2 cells, a human hepatocellular carcinoma cell line, induces the CYP1 family and Nrf2. RNAi for AhR abolishes the induction of expression of CYP1 and Nrf2. These inductions are accompanied by the translocation of AhR into the nucleus in response to low-glucose conditions. Endogenous compounds are recruited as AhR ligands to induce various gene expressions, and our present results suggest that an endogenous AhR ligand is produced under low-glucose conditions and that the role of AhR as a transcription factor is related to the low-glucose response. The recommended glucose concentration (4.5 g/L) in the medium for culture of HepG2 was used as the high-glucose concentration in this study. We adopted 1.0 g/L as the low-glucose condition for elucidation of mechanisms of the stress response. These results will be useful to understand the relationship between drug-metabolizing enzymes and mechanisms of the anti-stress response of tumor cells, and will also be useful for investigating preventive remedies against tumor angiogenesis.

Combination of the histone deacetylase inhibitor depsipeptide and 5-fluorouracil upregulates major histocompatibilitycomplex class II and p21 genes and activates caspase-3/7 in human colon cancer HCT-116 cells

Epigenetic anticancer drugs such as histone deacetylase (HDAC) inhibitors have been combined with existing anticancer drugs for synergistic or additive effects. In the present study, we found that a very low concentration of depsipeptide, an HDAC inhibitor, potentiated the antitumor activity of 5-fluorouracil (5-FU) in a human colon cancer cell model using HCT-116, HT29, and SW48 cells via the inhibition of colony formation ability or cellular viability. Exposure to a combination of 5-FU (1.75 µM) and 1 nM depsipeptide for 24 and 48 h resulted in a 3- to 4-fold increase in activated caspase-3/7, while 5-FU alone failed to activate caspase-3/7. Microarray and subsequent gene ontology analyses revealed that compared to 5-FU or depsipeptide alone, the combination treatment of 5-FU and depsipeptide upregulated genes related to cell death and the apoptotic process consistent with the inhibition of colony formation and caspase-3/7 activation. These analyses indicated marked upregulation of antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class (GO:0002504) and MHC protein complex (GO:0042611). Compared with vehicle controls, the cells treated with the combination of 5-FU and depsipeptide showed marked induction (3- to 8.5-fold) of expression of MHC class II genes, but not of MHC class I genes. Furthermore, our global analysis of gene expression, which was focused on genes involved in the molecular regulation of MHC class II genes, showed enhancement of pro-apoptotic PCAF and CIITA after the combination of 5-FU and depsipeptide. These results may indicate a closer relationship between elevation of MHC class II expression and cellular apoptosis induced by the combination of depsipeptide and 5-FU. To the best of our knowledge, this is the first study to report that the combination of 5-FU and depsipeptide induces human colon cancer cell apoptosis in a concerted manner with the induction of MHC class II gene expression.

VEGF expression is regulated by HIF-1α and ARNT in 3D KYSE-70, esophageal cancer cell spheroids

In 3D cultured cell systems, the cells form 3D spheroids that mimic cancer cell spheroids in vivo. Cancer cells form cell spheroids as they grow. The in vivo spheroids do not contain a vascular network; therefore, oxygen and nutrition supplies are insufficient. Specifically, the cells in the core region of the cluster are exposed to higher stress levels than the cells in the outer spheroid layer. As a result, the cells in the spheroid are exposed to low nutrition and hypoxia conditions. To overcome these shortages, angiogenesis is induced in cancer spheroids in vivo. Vascular endothelial growth factor (VEGF) is an important molecule involved in angiogenesis. VEGF is secreted by cancer cells in vivo in response to stress conditions such as hypoxia. VEGF expression in cancer cells is mediated by hypoxia‐inducible factor 1α (HIF1α), which accumulates in cancer cells during hypoxia. In this report, we show that VEGF expression is regulated by HIF1α and that VEGF is secreted to the outside of the spheroid in vitro. Several investigators have reported that HIF1α forms a protein–protein complex with aryl hydrocarbon receptor translocator (ARNT). We report here that not only HIF1α but also ARNT regulates VEGF expression in 3D cancer spheroids. Our results suggest the utility of the in vitro 3D cancer spheroid model for investigating angiogenesis in cancerous tissues.

The regulation mechanism of AhR activated by benzo[ a ]pyrene for CYP expression are different between 2D and 3D culture of human lung cancer cells

Most of cytochrome P450 (CYP) expressions are regulated by nuclear receptors. The regulation pathways of transcription are activated by binding of the ligand to the receptor. Many combination of CYPs and nuclear receptors in transcriptional regulation have been reported. However, we have reported that the combination changes depending on culture condition on the same type of cells. The regulation pathway of CYP1A expression is different between 2D monolayer cultured cells and 3D spheroids of human liver cancer cells. Aryl hydrocarbon receptor (AhR) is one of the transcription factors for CYP1A and CYP1B1 expression, and this pathway is important for inducing human lung cancer. CYP1B1 expression in human lung cancer cells are regulated by AhR in 2D and 3D cells. But CYP1A expression are not induced by AhR in 3D cells. As with liver cancer cells, the function of AhR in lung cancer cells is different between 2D cells and 3D spheroids. These results important for understanding relationship between AhR and CYP expression before and after cell neoplastic formation in human lung.

Strategic Perspectives on Improved Anti-Tumor Drug Effects inCombination with Clinically Equivalent or Lower Concentrations ofEpigenetic Modifiers, DNA Methyltransferase Inhibitors, and HistoneDeacetylase Inhibitors

The aim of this research is to facilitate the pursuit of improved chemotherapeutic drugs in combination with epigenetic modifiers. Both in vitro studies and a clinical study have described the combinations of DNA methyltransferase inhibitors with irinotecan and histone deacetylase inhibitors with 5-fluorouracil or gemcitabine to enhance their anti-cancer activities. The molecular mechanisms involved in the potentiation of anti-tumor activities were apoptosis regulation, cellular metabolism, DNA topoisomerase-I upregulation, cell-cell adhesion, regulation of transcription (DNA-templated), DNA repair, and the PI3K/AKT signaling pathway. More importantly, the priming effects and long-lasting effects induced by DNA methyltransferase inhibitors, when applied as a pretreatment, sensitized cancer cells to subsequent anti-cancer drug treatments. The combinations of 5-fluorouracil and gemcitabine with histone deacetylase inhibitors (depsipeptide and valproic acid, respectively), increased the expression of major histocompatibility complex class II, which may warrant further investigation for possible accurate biomarkers and therapeutic targets. As valproic acid downregulated histone deacetylase in patients recruited in a clinical phase I/II study, the activity of valproic acid may be associated with the enhanced anti-tumor activity in combination with 5-fluorouracil. This research provides a positive perspective on the combination therapy of anticancer drugs with epigenetic modifiers.

Additional file 4: Figure S4. of Analysis of DNA methylation landscape reveals the roles of DNA methylation in the regulation of drug metabolizing enzymes

Changes in the mRNA expression levels of DME genes in three hepatoma cell lines after DAC treatment. DME genes showing upregulation in at least one hepatoma cell line (greater that twofold change) following DAC treatment are listed. The fold change is indicated in each box, and genes with fold changes of more than two are indicated by closed boxes. The six DME genes exhibiting correlations between mRNA expression and DNA methylation status are shown in highlighted boxes. (PDF 37.9 KB)