Small molecule epigenetic modulators for enhancing recombinant antibody production in CHO cell cultures.

Small molecule epigenetic modulators that modify epigenetic states in cells are useful tools for regulating gene expression by inducing chromatin remodeling. To identify small molecule epigenetic modulators that enhance recombinant protein expression in Chinese hamster ovary (CHO) cells, we examined eight histone deacetylase inhibitors (iHDACs) and six DNA methyltransferase inhibitors as chemical additives in recombinant CHO (rCHO) cell cultures. Among these, a benzamide-based iHDAC, CI994, was the most effective in increasing monoclonal antibody (mAb) production. Despite suppressing cell growth, the addition of CI994 to mAb-expressing GSR cell cultures at 10 µM resulted in a 2.3-fold increase in maximum mAb concentration due to a 3.0-fold increase in specific mAb productivity (qmAb ). CI994 increased mAb messenger RNA levels and histone H3 acetylation in GSR cells, and chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed that CI994 significantly increased the histone H3 acetylation level at the cytomegalovirus promoter driving mAb gene expression, indicating that chromatin remodeling in the promoter region results in enhanced mAb gene transcription and qmAb . Similar beneficial effects of CI994 on mAb production were observed in mAb-expressing CS13-1.00 cells. Collectively, our findings indicate that CI994 increases mAb production in rCHO cell cultures by chromatin remodeling resulting from acetylation of histones in the mAb gene promoter.

DNA methyltransferase inhibitors modulate histone methylation: epigenetic crosstalk between H3K4me3 and DNA methylation during sperm differentiation.

The process of cytodifferentiation in spermatogenesis is governed by a unique genetic and molecular programme. In this context, accurate 'tuning' of the regulatory mechanisms involved in germ cells differentiation is required, as any error could have dramatic consequences on species survival and maintenance. To study the processes that govern the spatial-temporal expression of genes, as well as analyse transmission of epigenetic information to descendants, an integrated approach of genetics, biochemistry and cytology data is necessary. As information in the literature on interplay between DNA methylation and histone H3 lysine 4 trimethylation (H3K4me3) in the advanced stages of murine spermatogenesis is still scarce, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, at the cytological level using immunocytochemistry methodology. Our results revealed a particular distribution of H3K4me3 during sperm cell differentiation and highlighted an important role for regulation of DNA methylation in controlling histone methylation and chromatin remodelling during spermatogenesis.

Epigenetic DNA Methylation of EBI3 Modulates Human Interleukin-35 Formation via NFkB Signaling: A Promising Therapeutic Option in Ulcerative Colitis.

Ulcerative colitis (UC), a severe chronic disease with unclear etiology that is associated with increased risk for colorectal cancer, is accompanied by dysregulation of cytokines. Epstein-Barr virus-induced gene 3 (EBI3) encodes a subunit in the unique heterodimeric IL-12 cytokine family of either pro- or anti-inflammatory function. After having recently demonstrated that upregulation of EBI3 by histone acetylation alleviates disease symptoms in a dextran sulfate sodium (DSS)-treated mouse model of chronic colitis, we now aimed to examine a possible further epigenetic regulation of EBI3 by DNA methylation under inflammatory conditions. Treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC) and TNFα led to synergistic upregulation of EBI3 in human colon epithelial cells (HCEC). Use of different signaling pathway inhibitors indicated NFκB signaling was necessary and proportional to the synergistic EBI3 induction. MALDI-TOF/MS and HPLC-ESI-MS/MS analysis of DAC/TNFα-treated HCEC identified IL-12p35 as the most probable binding partner to form a functional protein. EBI3/IL-12p35 heterodimers (IL-35) induce their own gene upregulation, something that was indeed observed in HCEC cultured with media from previously DAC/TNFα-treated HCEC. These results suggest that under inflammatory and demethylating conditions the upregulation of EBI3 results in the formation of anti-inflammatory IL-35, which might be considered as a therapeutic target in colitis.

Hypomethylating Agents in Lymphoma.

OPINION STATEMENT: Epigenetic mutations are frequent and pathogenic in select subtypes of lymphoma, and agents modulating DNA and histone methylation-such as inhibitors of DNMT and EZH2, respectively-have demonstrated promise in treating these diseases. In particular, lymphomas derived from the germinal center-GC-DLBCL, FL, and AITL-are all characterized by epigenetic derangements. In an effort to target these derangements, DNMT inhibitors have been investigated as a means of improving responsiveness to chemotherapy in DLBCL patients, or as monotherapy or in combination with other epigenetic agents in the treatment of TCL. Histone methyltransferase inhibitors have demonstrated effectiveness in R/R FL patients with EZH2-activating mutations. New treatment options that target the pathogenesis of disease are needed. HDAC inhibitors have been in the clinic for over a decade for the treatment of lymphoma, and now methyltransferase inhibitors are finding their niche for this disease.

A phthalimidoalkanamide derived novel DNMT inhibitor enhanced radiosensitivity of A549 cells by inhibition of homologous recombination of DNA damage.

Purpose To elucidate the radiosensitizing effect and underlying mechanism of a new kind of DNA methyltransferase (DNMT) inhibitor with biological availability. Methods A novel non-nucleoside compound, designated as MA-17, was recently derived from a phthalimido alkanamide structure. DNMT expressions were confirmed in cultured human lung cancer (A549) and normal astrocyte (NHA) cells, radiosensitivity was measured using clonogenic assay, and assays of cell cycle alteration, apoptosis, DNA damage repair, and differential gene expression were undertaken. Results MA-17 significantly radiosensitized A549 cells with a mean dose enhancement ratio (DER) of 1.43 at the surviving fraction of 0.2 (p < 0.05 by one-tailed ratio paired t-test). MA-17 did not affect normal astrocytes (mean DER0.2, 1.016; p = 0.420). MA-17 demonstrated a mean half-life of 1.0 h in vivo and a relatively even distribution in various tissues. Pretreatment with MA-17 increased sub-G1 fractions and inhibited the repair of DNA double-strand breaks, which are induced by irradiation. We found that MA-17 also down-regulated DNA homologous recombination and the Fanconi anemia pathway (FANCA, BRCA1, and RAD51C) in A549 cells. This bioinformatics finding was confirmed in validation Western blot to evaluate the expression of vital proteins. Conclusions A novel phthalimido alkanamide derivative, a DNMT inhibitor, possessed both biostability and favorable and substantial radiosensitizing effects by augmenting apoptosis or inhibiting DNA damage repair.

Pharmacological inhibition of DNA methylation attenuates pressure overload-induced cardiac hypertrophy in rats.

BACKGROUND: Heart failure is associated with altered gene expression and DNA methylation. De novo DNA methylation is associated with gene silencing, but its role in cardiac pathology remains incompletely understood. We hypothesized that inhibition of DNA methyltransferases (DNMT) might prevent the deregulation of gene expression and the deterioration of cardiac function under pressure overload (PO). To test this hypothesis, we evaluated a DNMT inhibitor in PO in rats and analysed DNA methylation in cardiomyocytes. METHODS AND RESULTS: Young male Wistar rats were subjected to PO by transverse aortic constriction (TAC) or to sham surgery. Rats from both groups received solvent or 12.5 mg/kg body weight of the non-nucleosidic DNMT inhibitor RG108, initiated on the day of the intervention. After 4 weeks, we analysed cardiac function by MRI, fibrosis with Sirius Red staining, gene expression by RNA sequencing and qPCR, and DNA methylation by reduced representation bisulphite sequencing (RRBS). RG108 attenuated the ~70% increase in heart weight/body weight ratio of TAC over sham to 47% over sham, partially rescued reduced contractility, diminished the fibrotic response and the downregulation of a set of genes including Atp2a2 (SERCA2a) and Adrb1 (beta1-adrenoceptor). RG108 was associated with significantly lower global DNA methylation in cardiomyocytes by ~2%. The differentially methylated pathways were "cardiac hypertrophy", "cell death" and "xenobiotic metabolism signalling". Among these, "cardiac hypertrophy" was associated with significant methylation differences in the group comparison sham vs. TAC, but not significant between sham+RG108 and TAC+RG108 treatment, suggesting that RG108 partially prevented differential methylation. However, when comparing TAC and TAC+RG108, the pathway cardiac hypertrophy was not significantly differentially methylated. CONCLUSIONS: DNMT inhibitor treatment is associated with attenuation of cardiac hypertrophy and moderate changes in cardiomyocyte DNA methylation. The potential mechanistic link between these two effects and the role of non-myocytes need further clarification.

Anti-senescence effects of DNA methyltransferase inhibitor RG108 in human bone marrow mesenchymal stromal cells.

Alteration of DNA methylation is highly associated with ageing and ageing-related diseases. Remedy of the altered methylation pattern may provide beneficial efficacy in these diseases. In this study, we used a DNA methyltransferase inhibitor, RG108, to investigate the senescence effects in human bone marrow mesenchymal stromal cells (hBM-MSCs). First, we determined the optimized dose and time of RG108 treatment in hBM-MSCs to be 5 µM for 48 H, respectively. Under these conditions, the anti-senescence genes TERT, bFGF, VEGF, and ANG were increased, whereas the senescence-related genes ATM, p21, and p53 were decreased. The number of ß-galactosidase-positive cells was significantly decreased in RG108-treated MSCs, whereas the rates of MSC migration and cellular protection were increased. We have shown that RG108 significantly induces the expression of TERT by blocking methylation at the TERT promoter region. Thus, these data indicate that an optimized dose of RG108 may improve the cell migration, protection, cellular senescence, which may provide a better efficacy of these cells in stem cell therapy.

Therapeutic targeting of DNA methylation alterations in cancer.

DNA methylation is a critical component of gene regulation and plays an important role in the development of cancer. Hypermethylation of tumor suppressor genes and silencing of DNA repair pathways facilitate uncontrolled cell growth and synergize with oncogenic mutations to perpetuate cancer phenotypes. Additionally, aberrant DNA methylation hinders immune responses crucial for antitumor immunity. Thus, inhibiting dysregulated DNA methylation is a promising cancer therapy. Pharmacologic inhibition of DNA methylation reactivates silenced tumor suppressors and bolster immune responses through induction of viral mimicry. Now, with the advent of immunotherapies and discovery of the immune-modulatory effects of DNA methylation inhibitors, there is great interest in understanding how targeting DNA methylation in combination with other therapies can enhance antitumor immunity. Here, we describe the role of aberrant DNA methylation in cancer and mechanisms by which it promotes tumorigenesis and modulates immune responses. Finally, we review the initial discoveries and ongoing efforts to target DNA methylation as a cancer therapeutic.

Decitabine improves MMS-induced retinal photoreceptor cell damage by targeting DNMT3A and DNMT3B.

Introduction: Retinitis pigmentosa (RP) is a group of neurodegenerative retinopathies causing blindness due to progressive and irreversible photoreceptor cell death. The alkylating agent methyl methanesulfonate (MMS) can induce selective photoreceptor cell death, which is used to establish RP animal models. MMS induces DNA base damage by adding alkyl groups to DNA, and epigenetic modifications influence DNA damage response. Here, we aimed to explore the relationship between DNA methylation and DNA damage response in dying photoreceptors of RP. Methods: The mouse RP model was established by a single intraperitoneal injection of MMS. The retinal structure and function were assessed by H&E, OCT, TUNEL, and ERG at several time points. The expression of DNA methylation regulators was assessed by qPCR and Western blot. DNMT inhibitor 5-aza-dC was applied to inhibit the activity of DNA methyltransferases and improve the retinal photoreceptor damage. Results: The outer nuclear layer (ONL) and IS/OS layer were significantly thinner and the retinal function was impaired after MMS treatment. The cell death was mainly located in the ONL. The retinal damage induced by MMS was accompanied by hyperexpression of DNMT3A/3B. The application of DNMT inhibitor 5-aza-dC could suppress the expression level of DNMT3A/3B, resulting in the remission of MMS-induced photoreceptor cell damage. The ONL and IS/OS layers were thicker than that of the control group, and the retinal function was partially restored. This protective effect of 5-aza-dC was associated with the down-regulated expression of DNMT3A/3B. Conclusion: These findings identified a functional role of DNMT3A/3B in MMS-induced photoreceptor cell damage and provided novel evidence to support DNMTs as potential therapeutic targets in retinal degenerative diseases.Graphical Abstract.

Hydralazine and Panobinostat Attenuate Malignant Properties of Prostate Cancer Cell Lines.

Among the well-established alterations contributing to prostate cancer (PCa) pathogenesis, epigenetics is an important player in its development and aggressive disease state. Moreover, since no curative therapies are available for advanced stage disease, there is an urgent need for novel therapeutic strategies targeting this subset of patients. Thus, we aimed to evaluate the combined antineoplastic effects of DNA methylation inhibitor hydralazine and histone deacetylase inhibitors panobinostat and valproic acid in several prostate cell lines. The effect of these drugs was assessed in four PCa (LNCaP, 22Rv1, DU145 and PC-3) cell lines, as well as in non-malignant epithelial (RWPE-1) and stromal (WPMY-1) cell lines, using several assays to evaluate cell viability, apoptosis, proliferation, DNA damage and clonogenic potential. We found that exposure to each epidrug separately reduced viability of all PCa cells in a dose-dependent manner and that combined treatments led to synergic growth inhibitory effects, impacting also on colony formation, invasion, apoptotic and proliferation rates. Interestingly, antitumoral effects of combined treatment were particularly expressive in DU145 cells. We concluded that hydralazine and panobinostat attenuate malignant properties of PCa cells, constituting a potential therapeutic tool to counteract PCa progression.

Epigenetic modification in alcohol use disorder and alcoholic cardiomyopathy: From pathophysiology to therapeutic opportunities.

Alcohol consumption prompts detrimental psychological, pathophysiological and health issues, representing one of the major causes of death worldwide. Alcohol use disorder (AUD), which is characterized by compulsive alcohol intake and loss of control over alcohol usage, arises from a complex interplay between genetic and environmental factors. More importantly, long-term abuse of alcohol is often tied with unfavorable cardiac remodeling and contractile alterations, a cadre of cardiac responses collectively known as alcoholic cardiomyopathy (ACM). Recent evidence has denoted a pivotal role for ethanol-triggered epigenetic modifications, the interface between genome and environmental cues, in the organismal and cellular responses to ethanol exposure. To-date, three major epigenetic mechanisms (DNA methylation, histone modifications, and RNA-based mechanisms) have been identified for the onset and development of AUD and ACM. Importantly, these epigenetic changes induced by alcohol may be detectable in the blood, thus offering diagnostic, therapeutic, and prognostic promises of epigenetic markers for AUD and alcoholic complications. In addition, several epigenetic drugs have shown efficacies in the management of alcohol abuse, loss of control for alcohol usage, relapse, drinking-related anxiety and behavior in withdrawal. In this context, medications targeting epigenetic modifications may hold promises for pharmaceutical management of AUD and ACM.

Synthesis of novel 3-halo-3-nitroflavanones and their activities as DNA methyltransferase inhibitors in cancer cells.

The implication of DNA methylation in cancer is today clearly established. Despite that nucleoside analogues are currently used for leukaemia treatment, their low stability in physiological conditions and their lack of selectivity arise the need for the identification of non-nucleoside DNA methyltransferase inhibitors. Here, we describe the synthesis and pharmacological characterisation of a novel class of DNA methyltransferase inhibitors: the 3-halo-3-nitroflavanones. We showed that 3-bromo-3-nitroflavanones 3b and 4a have a micromolar DNMT inhibition and an increased potency in a cell reporter model. Interestingly they are significantly more stable than the reference compounds and induce a low cytotoxicity, supporting them as new candidates for the development of non-cytotoxic cell-reprogramming epi-drugs for anticancer treatment.

CBD modulates DNA methylation in the prefrontal cortex and hippocampus of mice exposed to forced swim.

Cannabidiol (CBD), a non-psychotomimetic component of Cannabis sativa plant, shows therapeutic potential in psychiatric disorders, including depression. The molecular mechanisms underlying the antidepressant-like effects of CBD are not yet understood. Previous studies in differentiated skin cells demonstrated that CBD regulates DNA methylation, an overall repressive epigenetic mechanism. Both stress exposure and antidepressant treatment can modulate DNA methylation in the brain, and lead to gene expression changes associated with depression neurobiology. We investigated herein if the antidepressant effect of CBD could be associated with changes in DNA methylation in the prefrontal cortex (PFC) and hippocampus (HPC) of mice submitted to the forced swimming test (FST). Therefore, we assessed: i) the behavioral effects induced by CBD and DNA methylation inhibitors (DNMTi: 5-AzaD and RG108), alone or in association; ii) the effects induced by CBD and DNMTi in global DNA methylation and DNMT activity, in PFC and HPC. Results showed that treatment with CBD (10 mg/kg), 5-AzaD and RG108 (0.2 mg/kg) induced an antidepressant-like effect in the FST. Similar effects were observed after the combination of sub-effective doses of CBD (7 mg/kg) and 5-AzaD or CBD (7 mg/kg) and RG108 (0.1 mg/kg). Also, stress reduced DNA methylation and DNMT activity in the HPC and increased it in the PFC. CBD and DNMTi treatment prevented these changes in both brain structures. Altogether, our results indicate that CBD regulates DNA methylation in brain regions relevant for depression neurobiology, suggesting that this mechanism could be related to CBD-induced antidepressant effects.

MicroRNAs: Diverse Mechanisms of Action and Their Potential Applications as Cancer Epi-Therapeutics.

Usually, miRNAs function post-transcriptionally, by base-pairing with the 3'UTR of target mRNAs, repressing protein synthesis in the cytoplasm. Furthermore, other regions including gene promoters, as well as coding and 5'UTR regions of mRNAs are able to interact with miRNAs. In recent years, miRNAs have emerged as important regulators of both translational and transcriptional programs. The expression of miRNA genes, similar to protein-coding genes, can be epigenetically regulated, in turn miRNA molecules (named epi-miRs) are able to regulate epigenetic enzymatic machinery. The most recent line of evidence indicates that miRNAs can influence physiological processes, such as embryonic development, cell proliferation, differentiation, and apoptosis as well as pathological processes (e.g., tumorigenesis) through epigenetic mechanisms. Some tumor types show repression of tumor-suppressor epi-miRs resulting in cancer progression and metastasis, hence these molecules have become novel therapeutic targets in the last few years. This review provides information about miRNAs involvement in the various levels of transcription and translation regulation, as well as discusses therapeutic potential of tumor-suppressor epi-miRs used in in vitro and in vivo anti-cancer therapy.

Epigenetics in hepatocellular carcinoma development and therapy: The tip of the iceberg.

Hepatocellular carcinoma (HCC) is a deadly tumour whose causative agents are generally well known, but whose pathogenesis remains poorly understood. Nevertheless, key genetic alterations are emerging from a heterogeneous molecular landscape, providing information on the tumorigenic process from initiation to progression. Among these molecular alterations, those that affect epigenetic processes are increasingly recognised as contributing to carcinogenesis from preneoplastic stages. The epigenetic machinery regulates gene expression through intertwined and partially characterised circuits involving chromatin remodelers, covalent DNA and histone modifications, and dedicated proteins reading these modifications. In this review, we summarise recent findings on HCC epigenetics, focusing mainly on changes in DNA and histone modifications and their carcinogenic implications. We also discuss the potential drugs that target epigenetic mechanisms for HCC treatment, either alone or in combination with current therapies, including immunotherapies.

Novel prodrugs of decitabine with greater metabolic stability and less toxicity.

BACKGROUND: DNA demethylation therapy is now used in practice for hematological tumors and is being developed for solid tumors. Nevertheless, it is difficult to achieve stable pharmacokinetics with the current DNA-demethylating agents, azacitidine (AZA) and decitabine (DAC), because of their rapid deamination by cytidine deaminase in vivo and spontaneous hydrolytic cleavage. Here, we aimed to develop metabolically stable prodrugs of AZA and DAC as novel DNA-demethylating agents. RESULTS: Thirty-five 5'-O-trialkylsilylated AZAs/DACs were synthesized with potential resistance to deamination. Out of these, 11 compounds exhibited demethylating activity similar to that of DAC and guadecitabine, and a suitable aqueous solubility. Pharmacokinetic analysis in mice showed that OR-2003 displayed the highest serum concentration and the area under the curve in an intraperitoneal experiment, whereas OR-2100 exhibited high stability to cytidine deaminase. Treatment of cells with OR-2003 and OR-2100 depleted DNA methyltransferase 1 completely and induced both gene-specific and genome-wide demethylation. The treatment suppressed the growth of multiple types of cancer cells and induced re-expression of tumor suppressor genes. The anti-tumor effect and DNA demethylation effect of OR-2003 and OR-2100 were comparable to that of DAC with fewer adverse effects in vivo. CONCLUSIONS: We developed two novel prodrugs of DAC that exhibited greater stability, comparable DNA demethylation activity, and less toxicity. These compounds are expected to overcome the difficulty in achieving stable pharmacokinetics in patients, leading to maximum DNA demethylation activity with minimum adverse effects.

Chemical screen for epigenetic barriers to single allele activation of Oct4.

Here we utilized the chromatin in vivo assay (CiA) mouse platform to directly examine the epigenetic barriers impeding the activation of the CiA:Oct4 allele in mouse embryonic fibroblasts (MEF)s when stimulated with a transcription factor. The CiA:Oct4 allele contains an engineered EGFP reporter replacing one copy of the Oct4 gene, with an upstream Gal4 array in the promoter that allows recruitment of chromatin modifying machinery. We stimulated gene activation of the CiA:Oct4 allele by binding a transcriptional activator to the Gal4 array. As with cellular reprograming, this process is inefficient with only a small percentage of the cells re-activating CiA:Oct4 after weeks. Epigenetic barriers to gene activation potentially come from heavy DNA methylation, histone deacetylation, chromatin compaction, and other posttranslational marks (PTM) at the differentiated CiA:Oct4 allele in MEFs. Using this platform, we performed a high-throughput chemical screen for compounds that increased the efficiency of activation. We found that Azacytidine and newer generation histone deacetylase (HDAC) inhibitors were the most efficient at facilitating directed transcriptional activation of this allele. We found one hit form our screen, Mocetinostat, improved iPSC generation under transcription factor reprogramming conditions. These results separate individual allele activation from whole cell reprograming and give new insights that will advance tissue engineering.

Identification of a novel quinoline-based DNA demethylating compound highly potent in cancer cells.

BACKGROUND: DNA methyltransferases (DNMTs) are epigenetic enzymes involved in embryonic development, cell differentiation, epithelial to mesenchymal transition, and control of gene expression, whose overexpression or enhanced catalytic activity has been widely reported in cancer initiation and progression. To date, two DNMT inhibitors (DNMTi), 5-azacytidine (5-AZA) and 5-aza-2'-deoxycytidine (DAC), are approved for the treatment of myelodysplastic syndromes and acute myeloid leukemia. Nevertheless, they are chemically instable and quite toxic for healthy cells; thus, the discovery of novel DNMTi is urgent. RESULTS: Here, we report the identification of a new quinoline-based molecule, MC3353, as a non-nucleoside inhibitor and downregulator of DNMT. This compound was able, in promoter demethylating assays, to induce enhanced green fluorescence protein (EGFP) gene expression in HCT116 cells and transcription in a cytomegalovirus (CMV) promoter-driven luciferase reporter system in KG-1 cells. Moreover, MC3353 displayed a strong antiproliferative activity when tested on HCT116 colon cancer cells after 48 h of treatment at 0.5 µM. At higher doses, this compound provided a cytotoxic effect in double DNMT knockout HCT116 cells. MC3353 was also screened on a different panel of cancer cells (KG-1 and U-937 acute myeloid leukemia, RAJI Burkitt's lymphoma, PC-3 prostate cancer, and MDA-MB-231 breast cancer), where it arrested cell proliferation and reduced viability after 48 h of treatment with IC50 values ranging from 0.3 to 0.9 µM. Compared to healthy cell models, MC3353 induced apoptosis (e.g., U-937 and KG-1 cells) or necrosis (e.g., RAJI cells) at lower concentrations. Importantly, together with the main DNMT3A enzyme inhibition, MC3353 was also able to downregulate the DNMT3A protein level in selected HCT116 and PC-3 cell lines. Additionally, this compound provided impairment of the epithelial-to-mesenchymal transition (EMT) by inducing E-cadherin while reducing matrix metalloproteinase (MMP2) mRNA and protein levels in PC-3 and HCT116 cells. Last, tested on a panel of primary osteosarcoma cell lines, MC3353 markedly inhibited cell growth with low single-digit micromolar IC50 ranging from 1.1 to 2.4 µM. Interestingly, in Saos-2 osteosarcoma cells, MC3353 induced both expression of genes and mineralized the matrix as evidence of osteosarcoma to osteoblast differentiation. CONCLUSIONS: The present work describes MC3353 as a novel DNMTi displaying a stronger in cell demethylating ability than both 5-AZA and DAC, providing re-activation of the silenced ubiquitin C-terminal hydrolase L1 (UCHL1) gene. MC3353 displayed dose- and time-dependent antiproliferative activity in several cancer cell types, inducing cell death and affecting EMT through E-cadherin and MMP2 modulation. In addition, this compound proved efficacy even in primary osteosarcoma cell models, through the modulation of genes involved in osteoblast differentiation.

Synthesis of 2-Amino-4-Fluoropyridine-C-Nucleoside Phosphoramidite for Incorporation into Oligonucleotides.

Straightforward and efficient methods for the synthesis of 2-amino-4-fluoropyridine-C-nucleoside (dF P) and the solid-phase synthesis of oligodeoxynucleotides containing dF P using a phosphoramidite are described. The synthesis of dF P is achieved by cross-coupling between a nucleobase (2-amino-4-fluoro-3,5-diiodopyridine) and sugar moieties. Its 3'-O-phosphoramidite is obtained by deiodination, 5'-O-protection, and 3'-O-phosphitilation in three steps. The phosphoramidite unit is compatible for the synthesis of oligonucleotides on solid-phase according to conventional phosphoramidite chemistry. The 2-amino-4-fluoropyridine-C-nucleoside moiety incorporated into the oligodeoxynucleotide reacts with a Cys residue in the catalytic site of DNA cytosine-5-methyltransferase (DNMT). It is apparent that 2-amino-4-fluoropyridine-C-nucleoside would be utilized in DNA-protein crosslink technology. This protocol describes the importance of solid-phase synthesis to obtain novel pyridine-C-nucleoside analogues and its incorporation into oligodeoxynucleotides in a short period of time. © 2019 by John Wiley & Sons, Inc.

DNA methylation profiles in cancer diagnosis and therapeutics.

Cancer initiation and proliferation is regulated by both epigenetic and genetic events with epigenetic modifications being increasingly identified as important targets for cancer research. DNA methylation catalyzed by DNA methyltransferases (DNMTs) is one of the essential epigenetic mechanisms that control cell proliferation, apoptosis, differentiation, cell cycle, and transformation in eukaryotes. Recent progress in epigenetics revealed a deeper understanding of the mechanisms of tumorigenesis and provided biomarkers for early detection, diagnosis, and prognosis in cancer patients. Although DNA methylation biomarker possesses potential contributing to precision medicine, there are still limitations to be overcome before it reaches clinical setting. Hence, the current status of DNA methylation biomarkers was reviewed and the future use in clinic was also predicted.