Current Pharmaceutical Design - Volume 20, Issue 11, 2014

In cancer cells, methylation-dependent gene silencing is at least partly mediated by the “Methyl-CpG-Binding Domain protein 2” (MBD2 protein), via the recruitment of chromatin remodeling complexes. However this repressive role was poorly investigated in normal cells. To identify the genes repressed by MBD2 in these cells, we have determined the impact of MBD2 depletion on gene expression in human embryonic MRC5 fibroblasts, using RNA inference combined with microarray analysis. The up-regulation of some randomly selected genes was confirmed and a direct association between gene repression and MBD2 binding on methylated promoters associated to these genes was subsequently established. This control of gene expression appears to depend on the CpG content of promoters as MBD2 depletion was not sufficient to induce the expression of silent genes associated with High-CpG promoters, but it was required to achieve the methyl-dependent transcriptional locking of the genes associated with promoters exhibiting intermediate CpG content. Therefore, MBD2 seems to play a selective role in gene repression depending on the CpG content of the promoter regions.

Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme for the salvage biosynthesis of nicotinamide adenine dinucleotide (NAD). Although elevated level of Nampt expression has been observed in various cancers, the involvement of Nampt promoter regulation was not well understood. We have identified a cluster of MEF2 recognition sites upstream of the functional hypoxia response elements (HREs) within the human Nampt promoter, and demonstrated that the two MEF2 sites at -1272 and -1200 were functional to upregulate the promoter activity by luciferase reporter assays. The Nampt promoter was able to be activated cooperatively following hypoxic stimulation by CoCl2 treatment with associated MEF2C overexpression. During the investigation on MEF2C regulation of endogenous Nampt expression in HeLa cells, the most significant enhancement of Nampt expression observed was by overexpression of MEF2C in combination with sodium butyrate exposure. By chromatin immunoprecipitation with a MEF2C anti-body, we found that MEF2C indeed interacted with endogenous Nampt promoter. The requirement of HDAC inhibition for the MEF2C enhancement of Nampt transcription was verified by RNAi of HDAC. Our results were in support of reports indicating that MEF2 family transcription factors interacted with HDACs and regulated downstream gene expression at the epigenetic levels. Our study provided important evidence to demonstrate the sophisticated mechanism of endogenous Nampt promoter regulation, and therefore, will help to better understand the Nampt overexpression in cancer progression, especially in the context of MEF2C upregulation which frequently occurred in cancer development and drug resistance.

Sir2-like proteins, known as sirtuins, have been under a spotlight in the realm of aging because of their positive effect on longevity in Saccharomyces.cerevisiae. Because Sir2 has been identified as a NAD+-dependent histone deacetylase, researchers have attributed its lifespan-extending utilities to gene silencing. Similar phenomena are found in multicellular eukaryotes by seemingly different mechanisms. In mammals, seven sirtuin homologs (SIRT1-7) have been identified, with varied cellular locations and molecular functions. Sirtuins target a wide spectrum of molecules for diversified post-translational modifications, thereby exerting multiple physiological benefits. The roles of sirtuins in cancer are still ambiguous, although they have been extensively studied. In this review, we summarize the multiple physiological roles played by sirtuins and their putative mechanisms, especially in cancer.

Epigenetic regulation is essential to the well-being of developing as well as developed cells by providing tissue-specific gene expression. DNA methylation on cytosine nucleotides is one of the core elements of epigenetic machinery, and stable DNA methylation patterns are maintained by properly regulated DNA methylation and DNA demethylation. DNA methylation has been studied extensively in the past 15 years, while the DNA demethylation process has largely been unknown. In this review, we briefly survey recent advances on DNA methylation/demethylation axis with special emphasis on the aspects of DNA demethylation.

Objectives: Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme for DNA biosynthesis and the epigenetic process of DNA methylation. MTHFR gene polymorphisms have been implicated as risk factors for several types of cancers. However, reports on the association of MTHFR polymorphisms with ovarian cancers are inconclusive. The aim of this study is to summarize on the reported data and meta-analytically investigate the relationship between the MTHFR C677T and A1298C polymorphism and the risk of ovarian cancer. Methods: We searched for all published articles indexed in MEDLINE (1950-2012), EMBASE (1974-2012), and CNKI (1994-2012). Case-control or cohort studies that relating to MTHFR polymorphism and ovarian cancer women were included and data were extracted independently by two reviewers. The search yielded 21 articles, from which 7 studies met the inclusion criteria. We performed a metaanalysis involving 3493 patients with ovarian cancer and 3863 controls with Review Manager 5.1 software. Odds ratio (OR) and 95% confidence intervals (CIs) were used to evaluate the ovarian cancer risk. Results: All the available data considered together, no association between the MTHFR C677T polymorphism and ovarian cancer risk was found in any genetic variations. However, in the subgroup analysis by ethnicity of Asian and Caucasian, MTHFR 677T was associated with significantly increased ovarian cancer risk among Asian [T allele vs. C allele: OR=1.50, 95% CI: 1.25-1.81, P<0.0001; CT + TT vs. CC (dominant model): OR=1.49, 95% CI: 1.18-1.88, P=0.0009; TT vs. CT + CC (recessive model): OR=2.33, 95% CI: 1.57-3.45, P<0.00001], while, there was no significant increased risk in Caucasian. As for MTHFR A1298C polymorphism, no marked association was found in either group of Caucasian population, while no data was available to analyze in Asian population. Conclusions: The C677T polymorphism of the MTHFR gene is associated with the susceptibility of ovarian cancer in Asian population, suggesting that TT genotype may serve as a risk factor of ovarian cancer among Asian but not Caucasians. In addition, there is no association between A1298C gene polymorphism and ovarian cancer, including Caucasian and Asian women.

DNA methyltransferase 1 (DNMT1) plays a significant role in maintaining DNA methylation. Aberrant DNA methylation is a recognized feature of human cancers and folate is directly involved in DNA methylation via one-carbon metabolism. Previous reports also have suggested that folate deficiency was associated with many cancers. The aim of the present study was to evaluate the effect of folate deficiency and aberrant expression of DNA methyltransferase 1 (DNMT1) on cervical cancerization. The expression of DNMT1 protein and mRNA and levels of serum folate were detected in 238 women with a diagnosis of normal cervix (NCn = 53), cervical intraepithelial neoplasia (CIN I, n = 52; CIN II/III, n = 53), and squamous cell carcinoma of the cervix (SCC; n = 80). In addition, the expression of DNMT1 protein and mRNA was measured in cervical cancer cells (Caski and C33A) treated by different concentration of folate. Serum folate levels decreased and expression levels of DNMT1 protein and mRNA increased gradually with progressive severity of the cervix lesions (P<0.001). It was found that folate was able to reduce the viability of Caski or C33A cell (r=0.978, P=0.002; r=0.984, P<0.001) and regulated aberrant expression of DNMT1 protein (r=-0.859, P=0.01; r=-0.914, P<0.001) and mRNA (r=-0.297, P=0.159; r=0.433, P=0.034) in vitro. Our findings indicated that the low-level of serum folate and high-expression of DNMT1 protein or mRNA was significantly associated with cervical carcinogenesis. Folate deficiency and aberrant expression of DNA methyltransferase 1 had additive effect on cervical cancerization. Folate supplement and recovery of aberrant DNA methylation status may offer a new strategy for prevention and therapy of cancers.

HOXA10 plays an important role in the body structure and development. Recently, patterns of deregulated HOX expression have been identified in various cancers. Meanwhile, WT1 is closely associated with the HOXA10 gene, which is an inducible transcription factor . We hypothesized that during the process of the ovarian cancer pathogenesis, the HOXA10 promoter CpG hypomethylation coupled with decreased WT1 expression could co-regulate HOXA10 expression. After the treatment of 5-Aza-dC, ovarian cancer cell lines(SKOV-3 and HEY) significantly increased HOXA10 expression. Transfection of HOXA10 siRNA with SKOV-3 and HEY ovarian cancer cell lines significantly downregulated HOXA10 expression but upregulated WT1 expression, compared to the control siRNA transfection. In ovarian cancer samples, the HOXA10 expression is significantly high. Nevertheless, the expression of HOXA10 is low in normal ones(P<0.05). We tried to use the method of methylation-specific PCR(MSP) to find the CpG sites, which are located in the scope of the WT1’s core-binding consensus(GCGG). In ovarian cancer samples, methylation prevalence was extremely high with the low expression of HOXA10. However, in the normal ovarian samples, the methylation prevalence was lower with the high expression of HOXA10(P<0. 05). In all samples of epithelial and normal ovarian samples collected, the WT1 expression with the promoter CpG hypomethylation of HOXA10 both contribute to the regulation of HOXA10 expression. That is to say, HOXA10 promoter methylationpositive combined with the WT1-positive result in the low expression of HOXA10. On the other hand, the HOXA10 expression was the highest in methylation-negative and WT1-negative samples.

Clinical screening criteria, such as young age of endometrial cancer diagnosis and family history of signature cancers, have traditionally been used to identify women with Lynch Syndrome, which is caused by mutation of a DNA mismatch repair gene. Immunohistochemistry and microsatellite instability analysis have evolved as important screening tools to evaluate endometrial cancer patients for Lynch Syndrome. A complicating factor is that 15-20% of sporadic endometrial cancers have immunohistochemical loss of the DNA mismatch repair protein MLH1 and high levels of microsatellite instability due to methylation of MLH1. The PCR-based MLH1 methylation assay potentially resolves this issue, yet many clinical laboratories do not perform this assay. The objective of this study was to determine if clinical and pathologic features help to distinguish sporadic endometrial carcinomas with MLH1 loss secondary to MLH1 methylation from Lynch Syndrome-associated endometrial carcinomas with MLH1 loss and absence of MLH1 methylation. Of 337 endometrial carcinomas examined, 54 had immunohistochemical loss of MLH1. 40/54 had MLH1 methylation and were designated as sporadic, while 14/54 lacked MLH1 methylation and were designated as Lynch Syndrome. Diabetes and deep myometrial invasion were associated with Lynch Syndrome; no other clinical or pathological variable distinguished the 2 groups. Combining Society of Gynecologic Oncology screening criteria with these 2 features accurately captured all Lynch Syndrome cases, but with low specificity. In summary, no single clinical/pathologic feature or screening criteria tool accurately identified all Lynch Syndrome-associated endometrial carcinomas, highlighting the importance of the MLH1 methylation assay in the clinical evaluation of these patients.

A growing body of evidence supports that DNA methylation-mediated silencing of tumor suppressor genes plays a significant role in cancer development. DNA methylatransferase (DNMT) is the enzyme catalyzing the methylation modification of cytosines in a CpG dinucleotide context. In humans, this reaction is highly selective for certain gene promoters and/or genomic DNA domains. Elucidation of the intracellular targeting mechanism by DNMT has become the key task for understanding epigenetic regulation in cancers. Unfortunately, no suitable method is available to explore this important cell function. This study focuses on the development of an efficient technique for measuring the intracellular, DNMT isoform-specific, and methylated gene-specific, DNA methylation alterations. The technique, designated IMA for Intracellular DNA Methylation Assay, takes advantage of covalent arresting of active DNMT molecules by aza-deoxycytidine (ADC), a modified cytosine homologue readily incorporated into genomic DNA at the cytosine position. The DNMT-DNA complex was isolated by a modified ETOH precipitation procedure to remove cellular proteins including free DNMT. Chromatin immunoprecipitation using DNMT isoform-specific antibody was subsequently performed to collect DNMT-bound DNA fragments. PCR amplification was used to detect and quantify the isolated gene fragment. Validation of the IMA was performed by manipulating the DNMT activity, by treating with antisense oligonucleotides against DNMT1 and DNMT3B, and repeating the IMA experiment. One of the main discoveries with this technique was the observation of DNMT3B maintenance methylation activity. This new technique can be applied to examine the dynamic DNMT-specific action on diversified methylation-silenced genes, in a variety of cell culture conditions.

Hematopoietic progenitors derived from human embryonic stem cells (hESCs) present both a potential cell source for cellreplacement therapies and an in vitro model for hematopoietic stem cell (HSC) development. Current protocols for the hematopoietic differentiation of hESCs suffer from low efficiency and functional defects in the derived HSCs. Epigenetic mechanisms of HSC development should be addressed to overcome these imperfections. The focus of this review is to summarize the knowledge on the epigenetic regulation of pluripotency and lineage-specific genes with the emphasis on the hematopoietic cell lineage. The potential utilization of this knowledge to improve the generation of HSCs for clinical application is also discussed.

Prostate cancer (PCa) is a very common neoplasm, which is generally treated by chemo-, radio-, and/or hormonal-therapy. After a variable time, PCa becomes resistant to conventional treatment, leading to patient death. Prostate tumor-initiating cells (TICs) and cancer repopulating cells (CRCs) are stem-like populations, driving respectively cancer initiation and progression. Histone modifiers (HMs) control gene expression in normal and cancer cells, thereby orchestrating key physiological and pathological processes. In particular, Polycomb group genes (PcGs) are a set of HMs crucial for lineage-specific gene silencing and stem cell self renewal. PcG products are organized into two main Polycomb Repressive Complexes (PRCs). At specific loci, PRC2 catalyzes histone H3 Lys27 trimethylation, which triggers gene silencing by recruiting PRC1, histone deacetylases and DNA methyl transferases. PRC1 catalyzes addition of the repressive mark histone H2A ubiquitination. Recently, the catalytic component of PRC1 (BMI1) was shown to play critical roles in prostate CRC self-renewal and resistance to chemotherapy, resulting in poorer prognosis. Similarly, pharmacological disruption of PRC2 by a small molecule inhibitor reduced the tumorigenicity and metastatic potential of prostate CRCs. Along with PcGs, some histone lysine demethylases (KDMs) are emerging as critical regulators of TIC/CRC biology. KDMs may be inhibited by specific small molecules, some of which display antitumor activity in PCa cells at micromolar concentrations. Since epigenetic gene regulation is crucial for stem cell biology, exploring the role of HMs in prostate cancer is a promising path that may lead to novel treatments.

While the epithelial-mesenchymal transition (EMT) plays a fundamental role during development, its deregulation can adversely promote tumor metastasis. The phenotypic and cellular plasticity of EMT indicates that it is subject to epigenetic regulation. A hallmark of EMT is E-cadherin suppression. In this review, we try to embrace recent findings on the transcription factor Snail-mediated epigenetic silencing of E-cadherin. Our studies as well as those of others independently demonstrated that Snail can recruit various epigenetic machineries to the E-cadherin promoter. Based on these results, we propose a model of epigenetic regulation of EMT governed by Snail. Briefly, recruitment of the LSD1/HDAC complex by Snail facilitates histone H3K4 demethylation and H3/H4 deacetylation. Histone deacetylation may promote subsequent recruitment of PRC2 to methylate H3K27, while H3K4 demethylation favors the association of H3K9 methyltransferases G9a and Suv39H1. Finally, DNA methyltransferases (DNMTs) can be recruited to the promoter area in a G9a/Suv39H1-dependent manner. Together, these chromatin-modifying enzymes function in a Snail-mediated, highly orchestrated fashion to suppress E-cadherin. Disruption of the connection between Snail and these epigenetic machineries may represent an efficient strategy for the treatment of EMT-related diseases, including tumor metastasis.

Nutrient utilization is dramatically altered when cells receive signals to proliferate. Characteristic metabolic changes enable cells to meet the large biosynthetic demands associated with cell growth and division. Changes in rate-limiting glycolytic enzymes redirect metabolism to support growth and proliferation. Metabolic reprogramming in cancer is controlled largely by oncogenic activation of signal transduction pathways and transcription factors. Although less well understood, epigenetic mechanisms may seem to contribute to the regulation of metabolic gene expression in cancer. Reciprocally, accumulating evidence suggests that metabolic alterations may affect the epigenome. Understanding the relation between metabolism and epigenetics in cancer cells may open new avenues for anti-cancer strategies. In multi-cellular systems, molecular signals promoting cell growth and proliferation mediate the switch between catabolism and anabolism. Both normal proliferating and cancer cells must achieve high levels of macromolecular biosynthesis to provide the raw materials needed to produce new daughter cells. From a therapeutic view point, it is of great interest to determine metabolic differences that exist between normal proliferating cells and cancer cells. Cancer cells also exhibit significant alterations in the epigenome. Recent data indicate that cellular metabolism and epigenetic phenomenon are engaged in crosstalk [1, 2]. Considering current efforts to target both cancer metabolism and epigenetics, an understanding of the relationship between these two key features is of paramount importance [3, 4]. Here we discuss the role of cellular metabolism in regulation of the epigenome. Moreover, we discuss how epigenetic changes may contribute to establish cancer-specific metabolic features.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were known to be risk factors for HCC, they were suspected to promote its development by eliciting epigenetic changes. However, the precise gene targets and underlying mechanisms have not been elucidated. Epigenetic regulation of gene expression has emerged as a fundamental aspect of cancer development and progression. The molecular mechanisms of carcinogenesis in hepatocellular carcinoma involve a complex interplay of both genetic and epigenetic factors. DNA methylation, post-translational modifications of histone proteins, chromatin remodeling, and noncoding RNAs are four major types of mechanistic layers in the field of epigenetics. HBV infection could affect methylation on p16INK4A, GSTP1, CDH1(E-cadherin), RASSF1A, p21WAF1/CIP1 genes, which may play important roles in the development of HCC. HCV infection was related to aberrant methylation on SOCS-1, Gadd45β, MGMT, STAT1 and APC. Other epigenetic alterations included histone proteins, chromatin remodeling, and noncoding RNAs were described in literature. Uncovering the epigenetic alterations of HBV/HCV-induced HCC carcinogenesis could highlight a new strategy for deciphering the mechanism of HCC tumorigenesis and development, as well as a potential diagnostic advantage.

DNA methylation is an important part of the epigenetic code governing gene expression. In human reproductive diseases, recent studies have shown the existence of deviations from the normal methylation profile at various genome loci. In this review, this type of epigenetic alterations is explored in pathological spermatogenesis, ovarian diseases, placental syndromes, such as preeclampsia and Intra- Uterine Growth Restriction, uterine diseases such as endometriosis, and putative pathophysiological effects of Assisted Reproductive Technologies. We review the notion of epigenetics, the technical methods available to analyze methylation, and the known associations between reproductive diseases and DNA methylation, focusing on human pathologies and on animal models when available. We show that imprinted genes control regions (ICRs) are a prominent and frequent target of methylation anomalies in reproductive disorders, but such alterations also affect non-imprinted genes. The mechanistic aspects of gene regulation in response to methylation anomalies are also discussed in this review when they have been investigated.

Genomic imprinting is among the most important epigenetic mechanisms whereby expression of a subset of genes is restricted to a single parental allele. Loss of imprinting (LOI) through hypo or hyper methylation is involved in various human syndromes. These LOI occur early during development and usually impair growth. Some imprinting syndromes are the consequences of genetic anomalies, such as uniparental disomies (UPD) or copy number variations (deletion or duplications) involving the imprinted domains; others are due to LOI at the imprinting control regions (ICR) regulating each domain. Imprinting disorders are phenotypically heterogeneous, although some share various common clinical features such that diagnosis may be difficult. Multilocus imprinting defects associated with several syndromes have been increasingly reported in recent years, although there are no obvious clinical differences between monolocus and multilocus LOI patients. Subsequently, some rare mutations of transacting factors have been identified in patients with multilocus imprinting defects but they do not explain the majority of the cases; this therefore implies that other factors are involved. By contrast, no mutation of a transacting factor has yet been identified in monolocus LOI. The effect of the environment on the regulation of imprinting is clearly illustrated by studies of assisted reproductive technology (ART). The regulation of imprinting is complex and involves a huge range of genetic and environmental factors; the identification of these factors will undoubtedly help to elucidate the regulation of imprinting and contribute to the understanding of imprinting disorders. This would be beneficial for diagnostics, clinical follow up and the development of treatment guidelines.

Worse reproductive health in the men born through intracytoplasmic sperm injection (ICSI) or other assisted reproductive techniques (ART) has been reported in many studies. However, owing to the interference of genetic and environmental factors, it is difficult to identify whether ICSI method would affect male reproductive health. Therefore, ART mouse models were established in this study. Besides semen quality, serum testosterone and histological analysis of testes, 6 paternally expressed imprinted genes were chosen to detect their expressions and methylation levels in testes of adult F1 and F2 mice. Although the phenotypic abnormalities weren’t found, Kcnq1ot1, Mest, Peg3, Plagl1 and Snrpn in ICSI group showed lower expressions than those in naturally conceived (NC) group. The expressions of Kcnq1ot1, Peg3 and Snrpn in in vitro fertilization (IVF) conceived mice was lower than those in controlled ovarian hyperstimulation (COH) conceived mice, but higher than those in ICSI mice. Most differences between NC and ICSI group and between IVF and ICSI group were also represented in F2 generations. During the methylation analysis, no matter there was significant difference between compared groups, the changing trends of methylation level were almost opposite to their corresponding gene expressions. These results indicated that the differential expressions of paternally expressed genes occurred in testes of ICSI mice, which may be mediated by methylation modification. Both ICSI procedure and mechanical stimulation can induce intergenerational transmission of the epigenetic changes. In vitro culture and mechanical stimulation were the main factors inducing the down regulation of paternally expressed imprinted genes in testes.

To investigate the possible mechanisms of the abnormal expression patterns of many genes in the embryos in vitro, the expression of histone acetyltransferase GCN5 and histone deacetylase 1 (HDAC1) were detected in mouse preimplantation embryos. For the in vitro group, the pronucleus embryos were obtained from superovulated mice, and cultured in vitro to get the two-cell, four-cell, eightcell, morula and blastocyst stages embryos. For the in vivo group, embryos at different stages were obtained from pregnant mice directly. Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were used to detect the mRNA and protein expression levels of GCN5 and HDAC1. Compared with the embryos in vivo, the expression levels of Gcn5 in the embryos in vitro significantly increased except those in four-cell embryos. The expression of Gcn5 in eight-cell embryos in vivo and in four-cell and eight-cell embryos in vitro was higher than those at other stages within the same group. Compared with the expressions of Hdac1 in embryo in vivo, only those at two-cell embryo in vitro showed decreased level. The expression of Hdac1 enhanced after two-cell embryo stage in vitro, but no difference showed in vivo. The protein expression of GCN5and HDAC1 in the embryo in vitro at every stage showed a lower level with the control of those in the embryos in vivo. Our studies indicated that in vitro culture could induce the expressed alteration of GCN5 and HDAC1, which might be related to the expression patterns of many other epigenetically regulated genes.

DNA methylation is an important regulatory component which influences phenotypes by modulating gene expression. Changes in DNA methylation may lead to altered phenotypes and ability of an organism to respond to stress leading to subsequent manifestation of life style diseases, cancer, etc. The human X chromosome represents a classical model for epigenetic processes governing differential regulation of homologous chromosomes. X monosomy (45, XO) leads to Turner's syndrome in human with mild to severe phenotypes. Using a novel cDNA based high throughput approach of assessing genome wide methylation; we have examined the methylation landscape in human fibroblasts in 45, XO and 46, XX individuals. We report here that as expected methylation of X linked genes is different in these two situations. It was observed that methylation of several autosomal genes is also affected in this X monosomy state. Genes involved in bone remodeling, glucose sensitivity and ovarian function appear to be altered in addition to genes involved in epigenetic regulatory processes. This opens up interesting possibility of misregulation of DNA methylation in the X monosomy state resulting in altered gene expression and altered phenotypes. This may be one of the reasons for the variance, differential severity and penetrance in case of Turner’s syndrome. We propose that a systematic analysis of the molecular genetic mechanisms governing this epigenetic regulation will open up new therapeutic interventions which will certainly help in reducing severity of the disease and help in better management of X monosomy (Turner’s syndrome).

Syncytin-1 plays a critical role in the maintenance of normal pregnancy by mediating the formation of syncytiotrophoblasts through a fosugenic action. Encoded by the human endogenous retrovirus envelope gene HERV-W, syncytin-1 trophoblast-specific expression is controlled by epigenetic mechanisms. In non-placental tissues, the syncytin-1 gene is suppressed by hypermethylation in the LTR promoter region. Hypomethylated and activated syncytin-1 gene is found in placental trophoblast lineages and malignant cells. We here demonstrate that while syncytin-1 gene remains silenced in the eutopic endometrium from endometriotic patients, syncytin-1 mRNA and protein are detected in ectopic, endometriotic lesions; particularly the endometrioid glandular endothelial cells. LINE-1 COBRA assay and immunohistochemistry using the 5-MC-specific antibody did not detect any changes in global DNA methylation in the endometriotic tissues. However, results from COBRA and bisulfite sequencing indicated that the LTR region of the syncytin-1 promoter is hypomethylated in endometriotic tissues, highlighting the significance of DNA demethylation in syncytin-1 gene activation. Analysis of DNA methyltransferase 3B (DNMT3B) mRNA levels revealed that DNMT3B3, an isoform carrying methyltransferase activity, is downregulated; whereas DNMT3B7, the isoform without enzymatic activity, is upregulated in the endometriotic tissues, pointing to positive and negative regulatory functions, respectively, of these isoforms on syncytin-1 methylation. These results have provided the first evidence supporting the involvement of epigenetic mechanisms for syncytin-1 upregulation in endometriotic tissues. Considering recent findings on the nonfusogenic activity of syncytin-1, its expression in endometriotic tissues suggests that this multifunctional protein may be implicated in the pathogenesis and/or progression of endometriosis.

Syncytin-1 is a protein coded by a human endogenous retrovirus (HERV) gene of the HERV-W family (HERVWE1). Syncytin- 1 mediates formation of syncytiotrophoblasts through fusion of cytotrophoblasts, a hallmark of terminal differentiation of placental trophoblast linage. Syncytin-1 also possesses nonfusogenic functions and regulates cell cycle progression. While decreased syncytin-1 expression and syncytium deficiency are considered important pathological changes in preeclampsia, the molecular mechanism(s) underlying syncytin-1 downregulation remains unclear. In this study, we confirmed that expression levels of syncytin-1 mRNA and protein were significantly lower in preeclamptic placentas compared to normal controls. Human chorionic somatomammotropin expression, a marker for syncytium function, was also decreased in preeclamptic placentas. The mRNA levels of ASCT2, the syncytin-1 receptor involved in cell fusion process, and GCMa, a transcriptional factor known to regulate syncytin-1 expression, were not significantly altered. Methylation in the 5’LTR of syncytin-1 promoter was quantified by COBRA, methylation-specific PCR, and DNA sequencing. Results from all three assays indicated significantly hypermethylated syncytin-1 promoter in preeclamptic placentas compared to normal controls. Methylation levels were inversely correlated with syncytin-1 mRNA levels, suggesting that hypermethylation may lead to syncytin-1 downregulation. Further experiments indicated that DNMT1 and DNMT3B3 mRNA and protein levels were increased in preeclamptic placentas, suggesting that higher DNA methyltransferase activity may contribute to the hypermethylation of syncytin-1 in preeclamptic placentas. These results indicated that aberrant hypermethylation is involved in downregulation of syncytin-1, and epigenetic alterations may play a significant role in the development of preeclampsia.

Background: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. Methods: The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-β2, by quantitative methylation-specific PCR and RT-PCR, respectively. Results: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-β2 promoter hypermethylation levels, although mRNA reexpression was only attained for GSTP1 and APC. Conclusions: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.

DNA methylation plays an important role in epigenetics signaling, having an impact on gene regulation, chromatin structure and development. Within the family of de novo DNA methyltransferases two active enzymes, DNMT3A and DNMT3B, are responsible for the establishment of the proper cytosine methylation profile during development. Defects in DNMT3s function correlate with pathogenesis and progression of monogenic diseases and cancers. Among monogenic diseases, Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is the only Mendelian disorder associated with DNMT3B mutations and DNA methylation defects of satellite and non-satellite regions. Similar CpG hypomethylation of the repetitive elements and gene-specific hypermethylation are observed in many types of cancer. DNA hyper-methylation sites provide targets for the epigenetic therapy. Generally, we can distinguish two groups of epi-drugs affecting DNMTs activity, i) nucleoside inhibitors, covalently trapping the enzymes, and bringing higher cytotoxic effect and (ii) nonnucleoside inhibitors, which block their active sites, showing less side-effects. Moreover, combining drugs targeting chromatin and those targeting DNA methylation enhances the efficacy of the therapy and gives more chances of patient recovery. However, development of more specific and effective epigenetic therapies requires more complete understanding of epigenomic landscapes. Here, we give an overview of the recent findings in the epigenomics field, focusing on those related to DNA methylation defects in disease pathogenesis and therapy.

Epigenetic modulation captures the lack of correlation between the genotype and the phenotype. It also provides an interface between environment and the genotype leading to functional plasticity of the genome. While drug response can be modulated by the epigenome, the therapeutic intervention by drugs can also be considered as an environmental cue for epigenetic alterations. The effect of genetic polymorphism has accrued considerable interest and population polymorphism leading to variation in drug response is being studied extensively. The available data on the epigenetic marking of the whole genome in different contexts implies that no biological pathway or process in the mammalian system is free of epigenetic influence and thus, drug metabolism would not be an exception. In the light of the fact that the epigenome is not only variable between individuals, but that it also varies between different tissues of the same individual and with the age of the individual, it is still a long journey to transit from the correlation to causal relationship between drug response and the epigenomic variations. The present review is focused on recent developments in the area and a brief discussion of the future prospects and challenges.

The neuroepigenome, i.e., the epigenome of the nervous system, has become interesting for therapeutics in the last years due to widespread availability of dedicated drugs. A pivotal role for neuroepigenetics is certainly implied, both in physiology and pathology, by the highly dynamic structural and functional rearrangements that constantly occur into the nervous system, globally known as plasticity. Moreover, the idea that the pathophysiology of several neuropsychiatric disorders might involve epigenetic mechanisms is increasingly taking place due to accumulating experimental data and by the evidence of a synergistic interaction between genes and environment beneath most sporadic forms of these diseases. In this paper we will review the available evidence on the use of epigenome-modifying drugs in the field of neuropsychiatry, shortly describing for each disease the underlying assumptions of an epigenetic dysregulation.

Cognitive disorders are an important group of disorders affecting the brain for which currently used drugs are often of low efficacy and mainly of symptomatic value. There is increasing evidence suggesting that epigenetic changes in gene expression underlie cognitive disorders. Advances in epigenetics have given rise to a new class of drugs, epigenetic drugs, that reverse epigenetic changes in gene expression. At present most work on epigenetic drugs focuses on two types of drugs: histone deacetylase (HDAC) inhibitors, and drugs targeting DNA methylation. This article describes the role of epigenetic drugs in treating cognitive disorders, focusing on Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. Epigenetic drugs may improve the clinical management of patients with cognitive disorders.

Accumulating evidence suggested that epigenetic changes such as promoter-specific DNA hypermethylation and histone deacetylation cause tumor suppressor gene silencing and contribute to malignant transformation. Treatment of cancer cells with HDAC inhibitors can reactivate the expression of silenced genes, block the cell cycle, and induce cell apoptosis. In vitro experiments in cancer cell cultures and in vivo studies using mouse xynograft model have shown that HDAC inhibitors deliver potent anti-cancer effects. Clinical trials have led to approval of SAHA (Vorinostat) for treatment of lymphoma. Endometrial cancer (EC) is the most frequent malignancy in women’s reproductive tract. EC is known for extensive epigenetic alterations, including overexpression of HDAC and DNMT enzymes, and the frequent epigenetic silencing of DNA repair genes such as MLH1, tumor suppressor genes PTEN, and progesterone receptor, which suggests a potentially high sensitivity of this type of cancer to HDAC inhibitors. Indeed, studies from many laboratories using various models have shown that HDAC inhibitors are promising chemotherapy reagents for endometrial cancers. This review summarizes the results from these studies, with an emphasis to provide an update on the new findings from new drugs. Background information on HDAC expression in EC, and features of HDAC inhibitors are presented based on their relevance to our focused topic. The combined application of HDAC inhibitors with radiation therapy and other conventional therapeutic reagents are also discussed.

Epigenetic changes including DNA methylation, histone modifications, chromatin remodeling and microRNAs play critical roles in tumorigenesis and tumor development. Reversal of epigenetic changes sensitizes some tumor cells to radiation. DNMT-I enhances the response of tumor cells to radiotherapy. AZA demethylated promoters of genes related to ionizing radiation response, such as p16 and hMLH1. The genes expression of the p53, RASSF1, and DAPK gene families was increased by 5-aza-CdR, which induces G2-M phase arrest and increased apoptosis. HDAC-I has both anti-tumor activity and radiation sensitization activity. HDAC-I disrupts both DNA damage sensing and repair processes: HDAC-I disrupts the association between HDAC enzyme and DNA sensor proteins 53BP1 and ATM. HDAC-I changes the acetylation status of both proteins involved in homologous recombination (HR) repair pathway which include BRCA1, Rad51, and Rad50, and proteins involved in non-homologous end joining (NHEJ) repair pathway which include Ku70, and DNA-PK. HDACs are also implicated as essential components in the DNA repair process itself. Besides the radiosensitizing mechanism of intervention of DNA repair, other possible mechanisms including cell cycle redistribution, acetylation of Hsp90, increased apoptosis, and decreased survival signals are also suggested. Some miRNAs also regulate the radiosensitivity of tumor cells. Inhibition of miR-34 expression or function, downregulation of miR-155, upregulation of miR-18a, Overexpression let-7g or knocking down LIN28B, and ectopically overexpressed miR-10 in cells with low endogenous miR-101 level increase the response of cells to irradiation. For radiation-resistant cancer cells, miR-7 sensitizes the radiation for cells which activated EGFR-PI3K-AKT signaling pathway;

Extensive evidence suggests that dysregulation of histone lysine acetylation is intimately linked with the development of cancer in epigenetic level. Histone acetylation on lysine is regulated mainly by the "pencil" ---Histone acetyltransferases (HATs) and the "eraser" –Histone deacetylases HDACs. Dramatic elevation of global histone deacetylation is considered as a biomarker for cancer. Therefore, current antitumor drug design often targets HDACs, inhibiting overexpressed HDAC in tumor cells with natural or synthesized small molecules like largazole. Recently, a novel largazole derivative (largazole-7) was designed and prepared by replacement of Val 1 with tyrosine, and this modification increases selectivity toward human cancer cells over normal cells more than 100-fold. However, it is unclear about the dynamic level of histone acetylation under the treatment of this drug. It is also unclear whether the other modifications are also affected by largazole-7 treatment. Therefore, a global mapping of modifications on the histone proteins of cancer cell line treated by this drug may be of great benefit to elucidating its molecular mechanisms and exploring its potent as an antitumor drug. To realize the goal, we combined stable isotope labeling by amino acids in cell culture (SILAC) and high resolution MS for comprehensive identification and quantitative analysis of histone lysine acetylation and other modifications of Human Colon Cancer Cells (HCT-116) with and without treatment of largazole-7. In this analysis, we identified 68 histone PTMs in 38 sites on core histones, including lysine acetylation, methyaltion and butyrylation, a novel lysine modification. Further quantitative analysis not only discovered the global increased acetylated lysines, but also observed the changes of abundance of lysine methylation and butyrylation under stimulation of the drug. To our knowledge, it is the first report that regulation of largazole-7 against lysine butyrylation. Our study expands the catalog of histone marks in cancer, and provides an approach for understanding the known and new epigenetic marks under treatment of drugs.

Endometrial cancer, the most common gynecologic malignancy, is a hormonally-regulated tumor. Response to progestin-based therapy correlates positively with progesterone receptor (PR) expression. However, many endometrial tumors have low levels or loss of PR, limiting the clinical application of progestin. We evaluated the ability of epigenetic modulators to restore functional PR expression in Type I endometrial cancer cells with low basal PR. Treatment with the histone deacetylase inhibitor (HDACi) LBH589 induced a profound upregulation of PR mRNA. LBH589 restored PR protein expression at 24 hours and sustained expression for 72 hours, even in the presence of progesterone. LBH589 promoted a dose-dependent increase in PR protein levels, with an obvious increase with 10 nM LBH589. To investigate if the restored PR is functional as a transcription factor, we examined PR nuclear localization and expression of PRE- or Sp1-containing target genes. After treatment with LBH589 in the absence or presence of progesterone, PR nuclear expression was increased as demonstrated by Western blotting of nuclear fractions and immunostaining. Next, restored PR upregulated FoxO1, p21, and p27 and downregulated cyclin D1 in a ligand-dependent manner. Finally, LBH589 treatment induced cell cycle arrest in G1 that was further augmented by progesterone. Regulation of PR target genes was also achieved with other HDAC inhibitors, indicating that agents in this class work similarly with respect to PR. Our findings reveal that epigenetic modulators can restore endogenous functional PR expression in endometrial cancer cells and suggest that strategies to re-establish PR expression will resensitize endometrial tumors to progestin therapy.

DNA methyltransferase (DNMT) and histone deacetylase are key enzymes mediating the epigenetic regulation of gene expression. DNA hypermethylation and/or histone deacetylation in promoter regions is often associated with downregulation or silencing of transcription. Epigenetic silencing of tumor suppressor genes plays an important role in malignant transformation. DNMT and HDAC inhibitors induce DNA demethylation and histone acetylation, respectively, leading to reactivation of silenced genes, and dramatic morphological and functional changes in cancer cells. In this study, we have conducted a series of experiments to characterize the effects of epigenetic inhibitors in endometrial cancer cells. Using cell lines representing different stages of endometrioid cancers, we examined the impact of DNMT inhibitor, ADC, and HDAC inhibitor, TSA, on cell cycle and apoptosis. We found that while both reagents were capable of inhibiting cell proliferation and inducing cell apoptosis, TSA appeared to be a more potent apoptosis inducer, but with a smaller effect on cell cycle. On the other hand, ADC exhibited strong effects on cell cycle regulation, but had smaller impact on cell apoptosis. We subsequently confirmed the presence of a strong synergism between DNMT and HDAC inhibitors. Thus, ADC and TSA exhibited strong cytostatic and apoptotic effects in endometrial cancer cell lines and the combined application may deliver the highest response in the clinical setting.

Epithelial ovarian cancer (EOC) is the leading cause of death among gynecologic malignancies. Despite great efforts to improve early detection and optimize chemotherapeutic regimens, the 5-year survival rate is only 30% for patients presenting with late-stage ovarian cancer. The high mortality of this disease is due to late diagnosis in over 70% of ovarian cancer cases. A class of small noncoding RNAs, or microRNAs, was found to regulate gene expression at the post-transcriptional level. Some, but not all, of the data indicated that the miR-200 family was dysregulated in a variety of malignancies. In this study, we demonstrated that miR-200a and E-cadherin were significantly upregulated in EOC compared to benign epithelial ovarian cysts and normal ovarian tissues. However, further stratification of the subject indicated that the expression levels of miR-200a were significantly downregulated in late-stage (FIGO III+V) and grade 3 groups compared with early stage (FIGO I+II) and grade 1 to 2 groups. Similarly, relatively low levels of miR-200a were observed in the lymph compared to the node-negative group. E-cadherin expression was found to be absent in normal ovarian tissue and was frequently expressed in benign epithelial ovarian cysts, with absence or low levels observed in late-stage ovarian cancers. There was a significantly positive correlation between miR-200a and E-cadherin in EOC. The biphasic expression pattern suggested that miR-200a levels may serve as novel biomarkers for the early detection of EOC, and miR-200a and E-cadherin are candidate targets for the development of new treatment modalities against ovarian cancer.

Recently, a group of microRNAs (miRNAs), the miR-200 family (miR-200s) has been found to be deregulated in multiple types of cancers, in which this family of miRNAs was demonstrated to play a pivotal role in tumor initiation, maintenance, malignant metastasis and chemotherapy resistance. By targeting several central inducers of the epithelial-to-mesenchymal transition (EMT), e.g. ZEB1, ZEB2 and SLUG, miR-200s are currently recognized as master regulators of EMT, thereby suppressing cancer invasion and metastasis. The involvement of miR-200s in angiogenesis has also been reported, and they were found to directly target VEGF-A, FLT1/VEGFR1 and KDR/VEGFR2, three key components of the VEGF signaling pathway. Importantly, miR-200s also modulate the self-renewal ability of cancer stem cells by targeting BMI1 and SUZ12. Aberrant expression of miR-200s has been shown to confer chemoresistant properties to various kinds of cancers. Thus, miR-200s, by playing critical and pleiotropic roles in malignancies, are promising targets for cancer therapy. Notably, it has been shown that several types of natural agents and herbal extracts could be employed to manipulate the expression of miR-200s, making the targeting of miR-200s in cancer therapy more clinically attractive. Nevertheless, a very recent study reported a metastasis-promoting role of miR-200s in breast cancer; thus, careful assessment should be conducted before applying therapeutic interventions using miR-200s as treatment targets. In this review, we will focus on our emerging understanding of the roles of miR- 200s in cancer, specifically their therapeutic potential in treating cancer.

G-protein coupled receptor 4 (GPR4) is a G protein-coupled receptor (GPCR) activated by sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC). Later studies indicated that GPR4 can serve as a proton sensor. GPR4 has been known to play a critical role in the tube formation of vascular endothelial cells, and GPR4 overexpression is observed in various types of malignancies, suggesting its involvement in the cancer- related angiogenesis. In this study, we examined the GPR4 expression levels in blood vessels of ovarian cancer, and analyzed the relationship between GPR4 expression and the clinical and pathological characteristics of patients with epithelial ovarian carcinomas (EOC). Results from immunohistochemistry showed that GPR4 is detectable in the endothelium of vessels of both EOC and benign ovarian tumor tissue, but the expression levels were significantly increased in EOC. Moreover the increased expression is accompanied by a higher microvascular density (MVD) in EOC compared to that in the benign ovarian tumors. We demonstrated a positive correlation between GPR4 expression density and MVD in EOC, but not benign ovarian tumor tissues. Further analyses indicated that GPR4 expression and MVD in EOC were correlated to the status of lymph node metastasis and clinical stage, but not significantly correlated to the pathological classifications, histopathological grades, the amounts of ascites, status of peritoneal cytology, tumor sizes, or patients’ ages. These results suggested that GPR4 may play an important role in the development of EOC, and its overexpression might be required for the angiogenesis, tumor growth, and metastasis of EOC.

Reactive oxygen species (ROS) plays a key role in carcinogenesis by aberrantly inducing signaling networks that initiatiate tumorigenesis and stimulate tumor progression. MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate approximately 30% of the genes in a cell via degradation or translational inhibition of their target mRNAs. However, the effects of ROS on miRNAs expression and the role of miRNAs in ROS-mediated injury on carcinogenesis are uncertain. Using UV spectrophotometry and enzyme-linked immunosorbent assay (ELISA), we examined tissues from human gastric cancers and tissues adjascent to gastric cancer and normal gastric tissues and found that total anti-oxidation competence (T-AOC), superoxide dismutase (SOD) and catalase (CAT) concentrations were lower in gastric cancer patients compared to the control subjects, while the concentrations of DNA oxidative damage product 8-oxo-deoxyguanosine (8-OHdG) was higher. To determine the potential role of miRNA in gastric carcinogenesis, real-time quantitative polymerase chain reaction (QPCR) analysis was performed. We found that human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) mRNA and miR-21 expression were significantly upregulated in gastric cancer tissues than in the adjacent normal gastric tissues. Furthermore, the expression of programmed cell death 4 protein (PDCD4) in gastric cancer tissues was significantly lower than in adjacent normal gastric tissues. The expression of miR-21 and PDCD4 was highly correlated with the degree of differentiation, tumor staging, local lymphatic node metastasis and remote metastasis. Expression of miR-21 was negatively correlated with T-AOC, SOD and CAT, but positively correlated with 8-OHdG and hOGG1mRNA. In addition, the relative expression of PDCD4 was negatively correlated with miR-21. These results suggest that the defensive balance of oxidation and antioxidant system in patients with GC was impaired, resulting in enhanced oxidative tissue injury, which may directly contribute to gastric carcinogenesis. Thus we conclude that ROS promotes gastric carcinogenesis via upregulating miR-21 expression which in turn down-regulates the expression of PDCD4 in gastric cancer cells.