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Millions of people around the world are exposed to elevated levels of arsenic through food or drinking water. Epidemiological studies have linked chronic arsenic exposure to an increased risk of several cancers, cardiovascular disease, central nervous system neuropathies, and genotoxic as well as immunotoxic effects. In addition to the induction of oxidative stress and inhibition of DNA repair processes, epigenetic effects, including altered DNA methylation patterns resulting in aberrant gene expression, may contribute to carcinogenicity. However, the underlying mechanisms by which chronic micromolar concentrations of arsenite affect the methylation status of DNA are not fully understood. In this study, human HepG2 hepatocarcinoma cells were treated with 0.5–10 μM sodium arsenite for 24 h, 10, or 20 days. During these periods, the effects on global DNA methylation, cell cycle phase distribution, and gene expression were investigated. While no impact on DNA methylation was seen after short-term exposure, global hypomethylation was observed at both long-term exposure periods, with concomitant induction of the DNA methyltransferase genes DNMT1 and DNMT3B, while DNMT3A was slightly down-regulated. Pronounced time- and concentration-dependent effects were also seen in the case of genes involved in DNA damage response and repair, inflammation, oxidative stress response, and metal homeostasis. These results suggest that chronic low-dose arsenite exposure can lead to global hypomethylation. As an underlying mechanism, the consistent down-regulation of DNA methyltransferase genes could be excluded; alternatively, interactions at the protein level could play an important role.

The nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor crucial in regulating cellular homeostasis and apoptosis. The NRF2 gene has been implicated in various biological activities, including antioxidant, anti-inflammatory, and anticancer properties. NRF2 can be regulated genetically and epigenetically at the transcriptional, post-transcriptional, and translational levels. Although DNA methylation is one of the critical biological processes vital for gene expression, sometimes, anomalous methylation patterns result in the dysregulation of genes and consequent diseases and disorders. Several studies have reported promoter hypermethylation downregulated NRF2 expression and its downstream targets. In contrast to the unalterable nature of genetic patterns, epigenetic changes can be reversed, opening up new possibilities in developing therapies for various metabolic disorders and diseases. This review discusses the current state of the NRF2-mediated antioxidative and chemopreventive activities of several natural phytochemicals, including sulforaphane, resveratrol, curcumin, luteolin, corosolic acid, apigenin, and most other compounds that have been found to activate NRF2. This epigenetic reversal of hypermethylated NRF2 states provides new opportunities for research into dietary phytochemistry that affects the human epigenome and the possibility for cutting-edge approaches to target NRF2-mediated signaling to prevent chronic disorders.

The epigenetic silencing of tumor suppressor genes (TSGs) is critical in the development of chronic myeloid leukemia (CML). SHP-1 functions as a TSG and negatively regulates JAK/STAT signaling. Enhancement of SHP-1 expression by demethylation provides molecular targets for the treatment of various cancers. Thymoquinone (TQ), a constituent of Nigella sativa seeds, has shown anti-cancer activities in various cancers. However, TQs effect on methylation is not fully clear. Therefore, the aim of this study is to assess TQs ability to enhance the expression of SHP-1 through modifying DNA methylation in K562 CML cells. The activities of TQ on cell cycle progression and apoptosis were evaluated using a fluorometric-red cell cycle assay and Annexin V-FITC/PI, respectively. The methylation status of SHP-1 was studied by pyrosequencing analysis. The expression of SHP-1, TET2, WT1, DNMT1, DNMT3A, and DNMT3B was determined using RT-qPCR. The protein phosphorylation of STAT3, STAT5, and JAK2 was assessed using Jess Western analysis. TQ significantly downregulated the DNMT1 gene, DNMT3A gene, and DNMT3B gene and upregulated the WT1 gene and TET2 gene. This led to hypomethylation and restoration of SHP-1 expression, resulting in inhibition of JAK/STAT signaling, induction of apoptosis, and cell cycle arrest. The observed findings imply that TQ promotes apoptosis and cell cycle arrest in CML cells by inhibiting JAK/STAT signaling via restoration of the expression of JAK/STAT-negative regulator genes.

Versican (VCAN), also known as extracellular matrix proteoglycan 2, has been suggested as a potential biomarker in cancers. Previous research has found that VCAN is highly expressed in bladder cancer. However, its role in predicting outcomes for patients with upper urinary tract urothelial cancer (UTUC) is not well understood. In this study, we collected tissues from 10 patients with UTUC, including 6 with and 4 without lymphovascular invasion (LVI), a pathological feature that plays a significant role in determining metastasis. Results from RNA sequencing revealed that the most differentially expressed genes were involved in extracellular matrix organization. Using the TCGA database for clinical correlation, VCAN was identified as a target for study. A chromosome methylation assay showed that VCAN was hypomethylated in tumors with LVI. In our patient samples, VCAN expression was also found to be high in UTUC tumors with LVI. In vitro analysis showed that knocking down VCAN inhibited cell migration but not proliferation. A heatmap analysis also confirmed a significant correlation between VCAN and migration genes. Additionally, silencing VCAN increased the effectiveness of cisplatin, gemcitabine and epirubicin, thus providing potential opportunities for clinical application.

With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and improving treatments. Nevertheless, upcoming, augmented recommendations against PSA screening underline an escalating disproportion between the benefit and harm of current diagnosis/prognosis and application of radical treatment standards. Undoubtedly, new potent diagnostic and prognostic tools are urgently needed to alleviate this tensed situation. They should allow a more reliable early assessment of the upcoming threat, in order to enable applying timely adjusted and personalized therapy and monitoring. Here, we present a basic study on an epigenetic screening approach by Methylated DNA Immunoprecipitation (MeDIP). We identified genes associated with hypomethylated CpG islands in three PCa sample cohorts. By adjusting our computational biology analyses to focus on single CpG-enriched 60-nucleotide-long DNA probes, we revealed numerous consistently differential methylated DNA segments in PCa. They were associated among other genes with NOTCH3, CDK2AP1, KLK4, and ADAM15. These can be used for early discrimination, and might contribute to a new epigenetic tumor classification system of PCa. Our analysis shows that we can dissect short, differential methylated CpG-rich DNA fragments and combinations of them that are consistently present in all tumors. We name them tumor cell-specific differential methylated CpG dinucleotide signatures (TUMS).

Urothelial carcinoma includes upper urinary tract cancer (UTUC) and bladder cancer. Although nephroureterectomy is the standard treatment for UTUC, the recurrence rate is approximately half and the tumor is associated with poor prognoses. Metastases are the most devastating and lethal clinical situation in urothelial carcinoma. Despite its clinical importance, few potential diagnostic biomarkers are suitable for early UC detection. We compared high-stage/high-grade urothelial carcinoma tissues to adjacent normal urothelial tissues using methyl-CpG binding domain protein capture for genome-wide DNA methylation analysis. Based on our findings, inhibin βA (INHBA) might be associated with carcinogenesis and metastasis. Further, clinical UC specimens had significant INHBA hypomethylation based on pyrosequencing. INHBA was detected by real-time PCR and immunohistochemistry staining, and was found to be highly expressed in clinical tissues and cell lines of urothelial carcinoma. Further, INHBA depletion was found to significantly reduce BFTC-909 cell growth and migration by INHBA-specific small interfering RNA. Interestingly, a positive correlation was found between SMAD binding and extracellular structure organization with INHBA using gene set enrichment analysis and gene ontology analysis. Together, these results are the first evidence of INHBA promoter hypomethylation and INHBA overexpression in UTUC. INHBA may affect urothelial carcinoma migration by reorganizing the extracellular matrix through the SMAD pathway.

Epigenetic silencing of tumor suppressor genes (TSGs) plays an essential role in cancer pathogenesis, including acute myeloid leukemia (AML). All of SHP-1, SOCS-1, and SOCS-3 are TSGs that negatively regulate JAK/STAT signaling. Enhanced re-expression of TSGs through de-methylation represents a therapeutic target in several cancers. Thymoquinone (TQ) is a major component of Nigella sativa seeds with anticancer effects against several cancers. However, the effects of TQ on DNA methylation are not entirely understood. This study aimed to evaluate the ability of TQ to re-express SHP-1, SOCS-1, and SOCS-3 in MV4-11 AML cells through de-methylation. Cytotoxicity, apoptosis, and cell cycle assays were performed using WSTs-8 kit, Annexin V-FITC/PI apoptosis detection kit, and fluorometric-red cell cycle assay kit, respectively. The methylation of SHP-1, SOCS-1, and SOCS-3 was evaluated by pyrosequencing analysis. The expression of SHP-1, SOCS-1, SOCS-3, JAK2, STAT3, STAT5A, STAT5B, FLT3-ITD, DNMT1, DNMT3A, DNMT3B, TET2, and WT1 was assessed by RT-qPCR. The molecular docking of TQ to JAK2, STAT3, and STAT5 was evaluated. The results revealed that TQ significantly inhibited the growth of MV4-11 cells and induced apoptosis in a dose- and time-dependent manner. Interestingly, the results showed that TQ binds the active pocket of JAK2, STAT3, and STAT5 to inhibit their enzymatic activity and significantly enhances the re-expression of SHP-1 and SOCS-3 through de-methylation. In conclusion, TQ curbs MV4-11 cells by inhibiting the enzymatic activity of JAK/STAT signaling through hypomethylation and re-expression of JAK/STAT negative regulators and could be a promising therapeutic candidate for AML patients.

Ribosome-binding protein 1 (RRBP1) is a potential oncogene in several cancer types. However, the correlation between RRBP1 expression and the prognosis of patients with upper tract urothelial carcinoma (UTUC) remains unclear. In this study, we identified that RRBP1 is associated with carcinogenesis and metastasis in UTUC using a methylation profiling microarray. High correlations between RRBP1 and cancer stages, nodal metastasis status, molecular subtypes, and prognosis in bladder urothelial cancer (BLCA) were found. Aberrant DNA methylation in the gene body region of RRBP1 was determined in UTUC tissues by methylation-specific PCR. RRBP1 expression was significantly increased in UTUC tissues and cell lines, as determined by real-time PCR and immunohistochemistry. RRBP1 depletion significantly reduced BFTC909 cell growth induced by specific shRNA. On the other hand, molecular subtype analysis showed that the expression of RRBP1 was associated with genes related to cell proliferation, epithelial–mesenchymal transition, and basal markers. A patient-derived organoid model was established to analyze patients’ responses to different drugs. The expression of RRBP1 was related to chemoresistance. Taken together, these results provide the first evidence that RRBP1 gene body hypomethylation predicts RRBP1 high expression in UTUC. The data highlight the importance of RRBP1 in UTUC malignancy and chemotherapeutic tolerance.

Hypomethylation of H19 and IGF2 can cause Silver–Russell syndrome (SRS), a clinically and genetically heterogeneous condition characterized by intrauterine growth restriction, poor postnatal growth, relative macrocephaly, craniofacial abnormalities, body asymmetry, hypoglycemia and feeding difficulties. Isolated hypomethylation of IGF2 has been reported in single cases of SRS as well. Here, we report on a 19-month-old patient who presented with two episodes of hypoglycemic seizures. No intrauterine growth restriction was observed, the patient did not present with SRS-typical facial features, and postnatal growth in the first months of life was along the lower normal percentiles. Exome sequencing did not reveal any likely pathogenic variants explaining the phenotype; however, hypomethylation studies revealed isolated hypomethylation of IGF2, while the methylation of H19 appeared normal. Hypoglycemia responded well to growth hormone therapy, and the boy showed good catch-up growth. Our case demonstrates that SRS and isolated IGF2 hypomethylation should be considered early in the diagnosis of recurrent hypoglycemia in childhood, especially in combination with small gestational age and poor growth.

Epigenetic regulation is a key component of stress responses, acclimatization and adaptation processes in plants. DNA methylation is a stable mark plausible for the inheritance of epigenetic traits, such that it is a potential scheme for plant breeding. However, the effect of modulators of stress responses, as hydrogen peroxide (H2O2), in the methylome status has not been elucidated. A transgenic tobacco model to the CchGLP gene displayed high H2O2 endogen levels correlated with biotic and abiotic stresses resistance. The present study aimed to determine the DNA methylation status changes in the transgenic model to obtain more information about the molecular mechanism involved in resistance phenotypes. The Whole-genome bisulfite sequencing analysis revealed a minimal impact of overall levels and distribution of methylation. A total of 9432 differential methylated sites were identified in distinct genome regions, most of them in CHG context, with a trend to hypomethylation. Of these, 1117 sites corresponded to genes, from which 83 were also differentially expressed in the plants. Several genes were associated with respiration, energy, and calcium signaling. The data obtained highlighted the relevance of the H2O2 in the homeostasis of the system in stress conditions, affecting at methylation level and suggesting an association of the H2O2 in the physiological adaptation to stress functional linkages may be regulated in part by DNA methylation.

The incidence and mortality rates of colorectal cancer (CRC) have been high in recent years. Prevention and early detection are crucial for decreasing the death rate. Therefore, this study aims to characterize the alteration patterns of mothers against decapentaplegic homolog 3 (SMAD3) in patients with CRC and its applications in early detection by using a genome-wide methylation array to identify an aberrant hypomethylation site in the intron position of the SMAD3 gene. Quantitative methylation-specific polymerase chain reaction showed that hypomethylated SMAD3 occurred in 91.4% (501/548) of Taiwanese CRC tissues and 66.6% of benign tubular adenoma polyps. In addition, SMAD3 hypomethylation was observed in 94.7% of patients with CRC from The Cancer Genome Atlas dataset. A decrease in circulating cell-free methylation SMAD3 was detected in 70% of CRC patients but in only 20% of healthy individuals. SMAD3 mRNA expression was low in 42.9% of Taiwanese CRC tumor tissues but high in 29.4% of tumors compared with paired adjacent normal tissues. Hypomethylated SMAD3 was found in cancers of the digestive system, such as liver cancer, gastric cancer, and colorectal cancer, but not in breast cancer, endometrial cancer, and lung cancer. In conclusion, SMAD3 hypomethylation is a potential diagnostic marker for CRC in Western and Asian populations.

(1) Background: RX-3117 (fluorocyclopentenyl-cytosine) is a cytidine analog that inhibits DNA methyltransferase 1 (DNMT1). We investigated the mechanism and potential of RX-3117 as a demethylating agent in several in vitro models. (2) Methods: we used western blotting to measure expression of several proteins known to be down-regulated by DNA methylation: O6-methylguanine-DNA methyltransferase (MGMT) and the tumor-suppressor genes, p16 and E-cadherin. Transport of methotrexate (MTX) mediated by the proton-coupled folate transporter (PCFT) was used as a functional assay. (3) Results: RX-3117 treatment decreased total DNA-cytosine-methylation in A549 non-small cell lung cancer (NSCLC) cells, and induced protein expression of MGMT, p16 and E-cadherin in A549 and SW1573 NSCLC cells. Leukemic CCRF-CEM cells and the MTX-resistant variant (CEM/MTX, with a deficient reduced folate carrier) have a very low expression of PCFT due to promoter hypermethylation. In CEM/MTX cells, pre-treatment with RX-3117 increased PCFT-mediated MTX uptake 8-fold, and in CEM cells 4-fold. With the reference hypomethylating agent 5-aza-2′-deoxycytidine similar values were obtained. RX-3117 also increased PCFT gene expression and PCFT protein. (4) Conclusion: RX-3117 down-regulates DNMT1, leading to hypomethylation of DNA. From the increased protein expression of tumor-suppressor genes and functional activation of PCFT, we concluded that RX-3117 might have induced hypomethylation of the promotor.

B-lymphocyte-induced maturation protein 1 (BLIMP1) exists as two major isoforms, α and β, which arise from alternate promoters. Inactivation of the full length BLIMP1α isoform is thought to contribute to B cell lymphomagenesis by blocking post-germinal centre (GC) B cell differentiation. In contrast, the shorter β isoform is functionally impaired and over-expressed in several haematological malignancies, including diffuse large B cell lymphomas (DLBCL). We have studied the influence on BLIMP1β expression of the Epstein-Barr virus (EBV), a human herpesvirus that is implicated in the pathogenesis of several GC-derived lymphomas, including a subset of DLBCL and Hodgkin’s lymphoma (HL). We show that BLIMP1β expression is increased following the EBV infection of normal human tonsillar GC B cells. We also show that this change in expression is accompanied by hypomethylation of the BLIMP1β-specific promoter. Furthermore, we confirmed previous reports that the BLIMP1β promoter is hypomethylated in DLBCL cell lines and show for the first time that BLIMP1β is hypomethylated in the Hodgkin/Reed-Sternberg (HRS) cells of HL. Our results provide evidence in support of a role for BLIMP1β in the pathogenesis of EBV-associated B cell lymphomas.

Somatic embryogenesis (SE) is a widely studied process due to its biotechnological potential to generate large quantities of plants in short time frames and from different sources of explants. The success of SE depends on many factors, such as the nature of the explant, the microenvironment generated by in vitro culture conditions, and the regulation of gene expression, among others. Epigenetics has recently been identified as an important factor influencing SE outcome. DNA methylation is one of the most studied epigenetic mechanisms due to its essential role in gene expression, and its participation in SE is crucial. DNA methylation levels can be modified through the use of drugs such as 5-Azacytidine (5-AzaC), an inhibitor of DNA methylation, which has been used during SE protocols. The balance between hypomethylation and hypermethylation seems to be the key to SE success. Here, we discuss the most prominent recent research on the role of 5-AzaC in the regulation of DNA methylation, highlighting its importance during the SE process. Also, the molecular implications that this inhibitor might have for the increase or decrease in the embryogenic potential of various explants are reviewed.

Evidence from several fields of investigation lead to the hypothesis that the sulfur atom is involved in vitamin B12-dependent methyl group transfer. To compile the evidence, it is necessary to briefly review the following fields: methylation, the new field of sulfane sulfur/hydrogen sulfide (S°/H2S), hydrosulfide derivatives of cobalamins, autoxidation of hydrosulfide radical, radical S-adenosylmethionine methyl transfer (RSMT), and methionine synthase (MS). Then, new reaction mechanisms for B12-dependent methyl group transfer are proposed; the mechanisms are facile and overcome difficulties that existed in previously-accepted mechanisms. Finally, the theory is applied to the effect of S°/H2S in nerve tissue involving the “hypomethylation theory” that was proposed 50 years ago to explain the neuropathology resulting from deficiency of vitamin B12 or folic acid. The conclusions are consistent with emerging evidence that sulfane sulfur/hydrogen sulfide may be beneficial in treating Alzheimer’s disease.

Epigenetic regulation is a key component of stress responses, acclimatization and adaptation processes in plants. DNA methylation is a stable mark plausible for the inheritance of epigenetic traits, such that it is a potential scheme for plant breeding. However, the effect of modulators of stress responses, as hydrogen peroxide (H 2 O 2 ), in the methylome status has not been elucidated. A transgenic tobacco model to the CchGLP gene displayed high H 2 O 2 endogen levels correlated with biotic and abiotic stresses resistance. The present study aimed to determine the DNA methylation status changes in the transgenic model to obtain more information about the molecular mechanism involved in resistance phenotypes. The Whole-genome bisulfite sequencing analysis revealed a minimal impact of overall levels and distribution of methylation. A total of 9432 differential methylated sites were identified in distinct genome regions, most of them in CHG context, with a trend to hypomethylation. Of these, 1117 sites corresponded to genes, from which 83 were also differentially expressed in the plants. Several genes were associated with respiration, energy, and calcium signaling. The data obtained highlighted the relevance of the H 2 O 2 in the homeostasis of the system in stress conditions, affecting at methylation level and suggesting an association of the H 2 O 2 in the physiological adaptation to stress functional linkages may be regulated in part by DNA methylation.

Cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-regulated chloride channel, is critical for secretion and absorption across diverse epithelia. Mutations or absence of CFTR result in pathogeneses, including cancer. While CFTR has been proposed as a tumor suppressing gene in tumors of the intestine, lung, and breast cancers, its effects in head and neck cancer (HNC) have yet to be investigated. This study aimed to define expression patterns and epigenetic modifications of CFTR in HNC. CFTR was expressed in normal but not in HNC cells and tissues. Treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR) was associated with rescued expression of CFTR, whose function was confirmed by patch clamp technique. Further experiments demonstrated that CFTR CpG islands were hypermethylated in cancer cells and tissues and hypomethylated in normal cells and tissue. Our results suggest that CFTR epigenetic modifications are critical in both down-regulation and up-regulation of CFTR expression in HNC and normal cells respectively. We then investigated the impact of CFTR on expressions and functions of cancer-related genes. CFTR silencing was closely associated with changes to other cancer-related genes, suppressing apoptosis while enhancing proliferation, cell motility, and invasion in HNC. Our findings demonstrate that hypermethylation of CFTR CpG islands and CFTR deficiency is closely related to HNC.