DNA Methyltransferase 1 Protein Expression Research Articles

Increased DNMT1 acetylation leads to global DNA methylation suppression in follicular granulosa cells during reproductive aging in mammals

With increasing age, the reproductive performance of women and female animals declines. However, the molecular mechanisms underlying ovarian aging and age-related fertility decline remain unclear. Granulosa cells (GCs) are suspected to play an important role in reproductive aging, and their proliferation, apoptosis, and steroid hormone secretion are used to determine the fate of follicles and ovarian function. First, we found that the proliferative ability of GCs from the old mouse group (10-month-old) decreased compared with that from the young mouse group (6-week-old), and cell cycle arrest occurred in old mice. To investigate changes in protein modification, we compared the levels of protein acetylation in GCs from young and old mice. We found that the K1118, K1120, K1122, and K1124 sites of DNA methyltransferase 1 (DNMT1) were increasingly acetylated with age, resulting in a decrease in DNMT1 protein expression. Therefore, we performed whole-genome methylation sequencing of GCs in the two groups and found that the CG methylation levels in the old group were lower than those in the young group. Furthermore, the inhibition of DNMT1 expression in GCs resulted in cell cycle arrest. This study revealed the dynamics and importance of protein acetylation and DNA methylation in GCs during reproductive aging. The findings provide a theoretical basis for studying the mechanism of reproductive aging in mammals.

Pirfenidone Reverts Global DNA Hypomethylation, Promoting DNMT1/UHRF/PCNA Coupling Complex in Experimental Hepatocarcinoma.

Hepatocellular carcinoma (HCC) development is associated with altered modifications in DNA methylation, changing transcriptional regulation. Emerging evidence indicates that DNA methyltransferase 1 (DNMT1) plays a key role in the carcinogenesis process. This study aimed to investigate how pirfenidone (PFD) modifies this pathway and the effect generated by the association between c-Myc expression and DNMT1 activation. Rats F344 were used for HCC development using 50 mg/kg of diethylnitrosamine (DEN) and 25 mg/kg of 2-Acetylaminofluorene (2-AAF). The HCC/PFD group received simultaneous doses of 300 mg/kg of PFD. All treatments lasted 12 weeks. On the other hand, HepG2 cells were used to evaluate the effects of PFD in restoring DNA methylation in the presence of the inhibitor 5-Aza. Histopathological, biochemical, immunohistochemical, and western blot analysis were carried out and our findings showed that PFD treatment reduced the amount and size of tumors along with decreased Glipican-3, β-catenin, and c-Myc expression in nuclear fractions. Also, this treatment improved lipid metabolism by modulating PPARγ and SREBP1 signaling. Interestingly, PFD augmented DNMT1 and DNMT3a protein expression, which restores global methylation, both in our in vivo and in vitro models. In conclusion, our results suggest that PFD could slow down HCC development by controlling DNA methylation.

Aberrant methylation and expression of TNXB promote chondrocyte apoptosis and extracullar matrix degradation in hemophilic arthropathy via AKT signaling.

Recurrent joint bleeding in hemophilia patients frequently causes hemophilic arthropathy (HA). Drastic degradation of cartilage is a major characteristic of HA, but its pathological mechanisms has not yet been clarified. In HA cartilages, we found server matrix degradation and increased expression of DNA methyltransferase proteins. We thus performed genome-wide DNA methylation analysis on human HA (N=5) and osteoarthritis (OA) (N=5) articular cartilages, and identified 1228 differentially methylated regions (DMRs) associated with HA. Functional enrichment analyses revealed the association between DMR genes (DMGs) and extracellular matrix (ECM) organization. Among these DMGs, Tenascin XB (TNXB) expression was down-regulated in human and mouse HA cartilages. The loss of Tnxb in F8-/- mouse cartilage provided a disease-promoting role in HA by augmenting cartilage degeneration and subchondral bone loss. Tnxb knockdown also promoted chondrocyte apoptosis and inhibited phosphorylation of AKT. Importantly, AKT agonist showed chondroprotective effects following Tnxb knockdown. Together, our findings indicate that exposure of cartilage to blood leads to alterations in DNA methylation, which is functionally related to ECM homeostasis, and further demonstrate a critical role of TNXB in HA cartilage degeneration by activating AKT signaling. These mechanistic insights allow development of potentially new strategies for HA cartilage protection.

Abstract 3245: Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia

Abstract Large granular lymphocyte (LGL) leukemia is a rare heterogeneous blood cancer that is characterized by the clonal lymphoproliferation of CD8+ T cells (T-LGL leukemia) or, less frequently, natural killer cells (NK-LGL leukemia). These cells evade activation induced cell death, allowing them to accumulate as highly cytotoxic lymphocytes that contribute to autoimmune attacks, neutropenia, and anemia. Frontline treatments are limited to repurposed, nonspecific immunosuppressive agents with limited efficacy and incomplete response rates. As there are no FDA-approved antineoplastic agents for LGL leukemia, our research explores novel therapeutic avenues for this orphan disease. Somatic activating mutations in the transcription factor STAT3 are the most common molecular alteration found in >50% of patients, and STAT3 signaling is dysregulated in almost all LGL leukemia patients. In the largest molecularly profiled LGL leukemia patient cohort to date, we found that STAT3 mutations frequently co-occur with mutations in chromatin and epigenetic modifying genes, suggesting crosstalk and cooperativity between STAT3 activation and epigenetic dysregulation. Moreover, TET2, which promotes DNA demethylation, is frequently inactivated in LGL leukemia via loss-of-function mutations and promoter hypermethylation. As DNA hypermethylation is an emerging hallmark of LGL leukemia, we evaluated the therapeutic potential of two epigenetic agents, azacitidine (AZA) and cladribine (CLAD), that directly and indirectly inhibit DNA hypermethylation, respectively. We hypothesize that AZA and CLAD will 1) exhibit cell intrinsic efficacy, 2) reprogram the epigenome of LGL leukemia through DNA hypomethylation to normalize aberrant gene expression, and 3) induce cytokine-mediated immune responses. AZA and CLAD cytotoxicity was assessed by the Cell Titer Glo viability assay in human LGL leukemia cell lines. Each exhibited potent anti-leukemic effects with IC50 values in the low micromolar and nanomolar ranges. Western blotting analysis demonstrated the decrease in protein expression of DNA methyltransferases and phosphorylated STAT3 levels with treatment in a time- and dose-dependent manner. We also explored the immunomodulatory effects of these hypomethylating agents since LGL leukemia is a pro-inflammatory malignancy with cytotoxic lymphocytes. Preliminary findings using the Isoplexis platform suggest that AZA and CLAD induce production of anti-inflammatory cytokines in LGL leukemia cells. Ongoing studies will identify differential methylation, chromatin accessibility, and gene expression using RRBS, ATAC-seq, and RNA-seq of AZA or CLAD treated samples to define the mechanisms and consequences of DNA demethylation. Uncovering the reprogramming capabilities of AZA and CLAD could allow for the potential repurposing of these inhibitors for treatment of LGL leukemia patients. Citation Format: Ariana Sabzevari, Ipsita Pal, Tess Deddens, Todd Fox, Aakrosh Ratan, David J. Feith, Thomas P. Loughran. Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3245.

Protective role of curcumin on broiler liver by modulating aflatoxin B1-induced DNA methylation and CYPs expression

The purpose of this study was to investigate the role of epigenetic DNA methylation and CYPs expression in AFB1-exposed broiler liver and the protective effect of curcumin. Sixty-four one-day-old AA broilers were randomly divided into four groups, including control group, AFB1 group (1 mg/kg AFB1), curcumin + AFB1 group (1 mg/kg curcumin) and curcumin group (300 mg/kg curcumin). Histological observation, CYP450 enzyme activities, the expression levels of DNA methyltransferases and CYP450 enzymes, and the overall DNA methylation level in broiler liver were investigated. Dietary AFB1 was found to induce severe liver injury in broilers, upregulate the mRNA and protein expression of CYP450 enzymes (CYP1A1, CYP1A2 and CYP3A4) and the enzyme activities of CYP1A2 and CYP3A4. According to HPLC, qPCR and western blot analyses, the overall DNA methylation level and the mRNA and protein expression of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) in the liver were significantly increased after AFB1 exposure. Importantly, the Pearson test and correlation analysis data revealed that the overall DNA methylation level of broiler liver was positively correlated with DNMTs, while CYP1A1, CYP1A2 and CYP3A4 were negatively correlated. Surprisingly, curcumin supplementation strongly ameliorated AFB1-induced hepatotoxicity by restoring the histological changes, decreasing the expression and enzymatic activity of liver CYP450 enzymes (CYP1A1, CYP1A2, and CYP3A4), and increasing the overall DNA methylation level and the expression of DNMTs. Taken together, we concluded that curcumin could protect against AFB1-induced liver injury by mediating the effects of DNA methylation and CYPs expression.

Melatonin: A Potential Regulator of DNA Methylation.

The pineal gland-derived indoleamine hormone, melatonin, regulates multiple cellular processes, ranging from chronobiology, proliferation, apoptosis, and oxidative damage to pigmentation, immune regulation, and mitochondrial metabolism. While melatonin is best known as a master regulator of the circadian rhythm, previous studies also have revealed connections between circadian cycle disruption and genomic instability, including epigenetic changes in the pattern of DNA methylation. For example, melatonin secretion is associated with differential circadian gene methylation in night shift workers and the regulation of genomic methylation during embryonic development, and there is accumulating evidence that melatonin can modify DNA methylation. Since the latter one impacts cancer initiation, and also, non-malignant diseases development, and that targeting DNA methylation has become a novel intervention target in clinical therapy, this review discusses the potential role of melatonin as an under-investigated candidate epigenetic regulator, namely by modulating DNA methylation via changes in mRNA and the protein expression of DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins. Furthermore, since melatonin may impact changes in the DNA methylation pattern, the authors of the review suggest its possible use in combination therapy with epigenetic drugs as a new anticancer strategy.

Prenatal BPA exposure disrupts male reproductive functions by interfering with DNA methylation and GDNF expression in the testes of male offspring rats.

BPA is a ubiquitous environmental endocrine-disrupting chemical, and maternal exposure to BPA is associated with impaired male reproductive functions; however, the mechanisms remain to be elucidated. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in maintaining normal spermatogenesis and fertility. However, the effect of prenatal BPA exposure on GDNF expression and its mechanism in the testis has not been reported. In this study, pregnant Sprague-Dawley rats were respectively exposed to 0, 0.05, 0.5, 5, and 50 mg/kg/day BPA via oral gavage from gestational day (GD) 5 to GD 19, with 6 pregnant rats in each group. ELISA, histochemistry, real-time PCR, western blot, and methylation-specific PCR (MSP) were used to detect the sex hormone levels, testicular histopathology, mRNA and protein expression of DNA methyltransferases (DNMTs) and GDNF, and the promoter methylation of Gdnf in the testes of male offspring at postnatal day (PND) 21 and PND 56. Prenatal BPA exposure increased body weight; decreased sperm counts and serum levels of testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH); and induced testicular histological damage, which indicated the damage of male reproductive function. Prenatal BPA exposure also upregulated Dnmt1 in 5 mg/kg group and Dnmt3b in 0.5 mg/kg group, but down-regulated Dnmt1 in 50 mg/kg group at PND 21. At PND 56, Dnmt1 was significantly increased in 0.05 mg/kg group but decreased in 0.5, 5, and 50 mg/kg groups, Dnmt3a was decreased, and Dnmt3b was markedly increased in 0.05 and 0.5 mg/kg groups but decreased in 5 and 50 mg/kg groups. The mRNA and protein expression levels of Gdnf were decreased markedly in 0.5 and 50 mg/kg groups at PND 21. And the methylation level of Gdnf promoter was significantly increased in 0.5 mg/kg group, but decreased in 5 and 50 mg/kg groups at PND 21. In conclusion, our study indicates that prenatal BPA exposure disrupts male reproductive functions, interferes with the expression of DNMTs, and decreases Gdnf expression in the testes of male offspring. Gdnf expression may be regulated by DNA methylation; however, the detailed mechanism needs to be further investigated.

Hsa_circ_0040809 regulates colorectal cancer development by upregulating methyltransferase DNMT1 via targeting miR-515-5p.

Circular RNAs (circRNAs) are key regulators in the progression of various cancers. Abnormal DNA methylation patterns feature prominently in the regulation of the expression of tumor-related genes. This study is aimed at investigating the molecular mechanism of circ_0040809 affecting colorectal cancer (CRC) progression by regulating DNA methyltransferase 1 (DNMT1). circ_0040809 was selected from the circRNA microarray datasets (GSE142837 and GSE138589). Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine the expression of circ_0040809, miR-515-5p, and DNMT1 mRNA in paired cancerous and paracancerous tissues of 40 CRC patients, as well as in cell lines. Western blotting was conducted for detecting DNMT1 protein expression in CRC cells. Cell proliferation, migration, and apoptosis were assessed through CCK-8, Transwell, and flow cytometry assays. Bioinformatics and dual-luciferase gene assay were conducted to predict and verify, respectively, the targeted relationships between circ_0040809 and miR-515-5p, as well as between miR-515-5p and DNMT1 mRNA. In CRC tissues and cells, circ_0040809 and DNMT1 expression are markedly increased, whereas miR-515-5p expression is decreased. Also, high circ_0040809 expression is significantly linked to shorter overall survival. Cell function compensation experiments reveal that circ_0040809 silencing inhibits CRC cell proliferation and migration and promotes apoptosis, while circ_0040809 overexpression has the opposite effects. Mechanistically, circ_0040809 competitively binds to miR-515-5p to elevate DNMT1 expression. Rescue assay reveals that overexpressed miR-515-5p partly counteracts the tumor-facilitating impact of circ_0040809. circ_0040809 facilitates CRC cell proliferation and migration, and inhibits apoptosis, through modulating miR-515-5p/DNMT1 axis. Our study implies that targeting circ_0040809 may be a therapy strategy for CRC treatment.

GCM2 Silencing in Parathyroid Adenoma Is Associated With Promoter Hypermethylation and Gain of Methylation on Histone 3.

Glial cells missing 2 (GCM2), a zinc finger-transcription factor, is essentially required for the development of the parathyroid glands. We sought to identify whether the epigenetic alterations in GCM2 transcription are involved in the pathogenesis of sporadic parathyroid adenoma. In addition, we examined the association between promoter methylation and histone modifications with disease indices. Messenger RNA (mRNA) and protein expression of GCM2 were analyzed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry in 33 adenomatous and 10 control parathyroid tissues. DNA methylation and histone methylation/acetylation of the GCM2 promoter were measured by bisulfite sequencing and chromatin immunoprecipitation-qPCR. Additionally, we investigated the role of epigenetic modifications on GCM2 and DNA methyltransferase 1 (DNMT1) expression in parathyroid (PTH)-C1 cells by treating with 5-aza-2'-deoxycytidine (DAC) and BRD4770 and assessed for GCM2 mRNA and DNMT1 protein levels. mRNA and protein expression of GCM2 were lower in sporadic adenomatous than in control parathyroid tissues. This reduction correlated with hypermethylation (P < .001) and higher H3K9me3 levels in the GCM2 promoter (P < .04) in adenomas. In PTH-C1 cells, DAC treatment resulted in increased GCM2 transcription and decreased DNMT1 protein expression, while cells treated with the BRD4770 showed reduced H3K9me3 levels but a nonsignificant change in GCM2 transcription. These findings suggest the concurrent association of promoter hypermethylation and higher H3K9me3 with the repression of GCM2 expression in parathyroid adenomas. Treatment with DAC restored GCM2 expression in PTH-C1 cells. Our results showed a possible epigenetic landscape in the tumorigenesis of parathyroid adenoma and also that DAC may be a promising avenue of research for parathyroid adenoma therapeutics.

Targeting Germ Cell Tumors with the Newly Synthesized Flavanone-Derived Compound MLo1302 Efficiently Reduces Tumor Cell Viability and Induces Apoptosis and Cell Cycle Arrest

Less toxic treatment strategies for testicular germ cell tumor (TGCT) patients are needed, as overtreatment is a concern due to the long-term side effects of platin-based chemotherapy. Although clinical benefit from classical hypomethylating agents has to date been limited, TGCTs show an abnormal DNA methylome indicating the potential of treating TGCTs with hypomethylating drugs. We tested, for the first time in TGCT cell lines, a new synthetic flavonoid compound (MLo1302) from the 3-nitroflavanone family of DNA methyltransferase (DNMT) inhibitors. We show that MLo1302 reduces cell viability (including of cisplatin resistant cell line NCCIT-R), with IC50s (inhibitory concentration 50) within the nanomolar range for NCCIT and NTERA-2 cells, and proved its cytotoxic effect. Exposure to MLo1302 reduced DNMT protein expression, similar to decitabine, and showed a partial effect in cell differentiation, reducing protein expression of pluripotency markers. RT2 profiler expression array indicated several dysregulated targets, related to activation of apoptosis, differentiation, and cell cycle arrest. We validated these data by showing increased apoptosis, increased protein expression of cleaved caspase 8 and activated caspase 2, and reduced proliferation (BrdU assay), with increase in CDKN1A and decrease in MIB-1 expression. Therefore, synthetic drugs designed to target DNA methylation in cells may uncover effective treatments for TGCT patients.

Stearoyl-CoA Desaturase 1 Activity Determines the Maintenance of DNMT1-Mediated DNA Methylation Patterns in Pancreatic β-Cells.

Metabolic stress, such as lipotoxicity, affects the DNA methylation profile in pancreatic β-cells and thus contributes to β-cell failure and the progression of type 2 diabetes (T2D). Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that is involved in monounsaturated fatty acid synthesis, which protects pancreatic β-cells against lipotoxicity. The present study found that SCD1 is also required for the establishment and maintenance of DNA methylation patterns in β-cells. We showed that SCD1 inhibition/deficiency caused DNA hypomethylation and changed the methyl group distribution within chromosomes in β-cells. Lower levels of DNA methylation in SCD1-deficient β-cells were followed by lower levels of DNA methyltransferase 1 (DNMT1). We also found that the downregulation of SCD1 in pancreatic β-cells led to the activation of adenosine monophosphate-activated protein kinase (AMPK) and an increase in the activity of the NAD-dependent deacetylase sirtuin-1 (SIRT1). Furthermore, the physical association between DNMT1 and SIRT1 stimulated the deacetylation of DNMT1 under conditions of SCD1 inhibition/downregulation, suggesting a mechanism by which SCD1 exerts control over DNMT1. We also found that SCD1-deficient β-cells that were treated with compound c, an inhibitor of AMPK, were characterized by higher levels of both global DNA methylation and DNMT1 protein expression compared with untreated cells. Therefore, we found that activation of the AMPK/SIRT1 signaling pathway mediates the effect of SCD1 inhibition/deficiency on DNA methylation status in pancreatic β-cells. Altogether, these findings suggest that SCD1 is a gatekeeper that protects β-cells against the lipid-derived loss of DNA methylation and provide mechanistic insights into the mechanism by which SCD1 regulates DNA methylation patterns in β-cells and T2D-relevant tissues.

Chlorogenic Acid Decreases Malignant Characteristics of Hepatocellular Carcinoma Cells by Inhibiting DNMT1 Expression.

BackgroundHepatocellular carcinoma (HCC) is the most common malignant tumor of the adult liver, exhibiting rapid progression and poor prognosis. Chlorogenic acid (CGA), a polyphenol, has several biological activities, including the suppression of liver cancer cell invasion and metastasis. Increased levels or alterations in the function of DNMT1 are associated with the inactivation of tumor suppressor genes. However, the CGA-affected DNMT1 expression mediated mechanism is still unclear.MethodsThe human hepatocellular carcinoma (HCC) HepG2 cells were treated with a positive control drug (5-AZA) or varying doses of CGA. DNA methyltransferase 1 (DNMT1) protein levels and other relevant proteins were evaluated using Western blotting and immunocytochemistry. Cell-cycle analysis was performed by flow cytometry-based PI staining, and cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The transwell invasion and wound healing assays were used to evaluate cell migration and invasion. In vivo proliferation of the HCC cells was detected. We investigated the expression of DNMT1, p53, p21, p-ERK, MMP-2, and MMP-9 in tumors using immunohistochemical analysis.ResultsOur results showed that CGA inhibited the proliferation, colony formation, invasion, and metastasis of HepG2 cells both in vitro and in vivo by down-regulating DNMT1 protein expression, which enhanced p53 and p21 activity, and resulting in a significant reduction in cell proliferation and metastasis. Moreover, CGA inactivated ERK1/2 and reduced MMP-2 and MMP-9 expression in HepG2 cells.ConclusionsCGA can suppress liver cancer cell proliferation, invasion, and metastasis through several pathways. CGA could serve as a candidate chemopreventive agent for HCC.

Age-dependent impairment of adipose-derived stem cells isolated from horses

BackgroundProgressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young (< 5), middle-aged (5–15), and old (> 15 years old).MethodsASCs were isolated from three age-matched horse groups using an enzymatic method. Molecular changes were assessed using qRT-PCR, ELISA and western blot methods, flow cytometry-based system, and confocal and scanning electron microscopy.ResultsOur findings showed that ASCs derived from the middle-aged and old groups exhibited a typical senescence phenotype, such as increased percentage of G1/G0-arrested cells, binucleation, enhanced β-galactosidase activity, and accumulation of γH2AX foci, as well as a reduction in cell proliferation. Moreover, aged ASCs were characterized by increased gene expression of pro-inflammatory cytokines and miRNAs (interleukin 8 (IL-8), IL-1β, tumor necrosis factor α (TNF-α), miR-203b-5p, and miR-16-5p), as well as apoptosis markers (p21, p53, caspase-3, caspase-9). In addition, our study revealed that the protein level of mitofusin 1 (MFN1) markedly decreased with increasing age. Aged ASCs also displayed a reduction in mRNA levels of genes involved in stem cell homeostasis and homing, like TET-3, TET-3 (TET family), and C-X-C chemokine receptor type 4 (CXCR4), as well as protein expression of DNA methyltransferase (DNMT1) and octamer transcription factor 3/4 (Oct 3/4). Furthermore, we observed a higher splicing ratio of XBP1 (X-box binding protein 1) mRNA, indicating elevated inositol-requiring enzyme 1 (IRE-1) activity and, consequently, increased endoplasmic reticulum (ER) stress. We also observed reduced levels of glucose transporter 4 (GLUT-4) and insulin receptor (INSR) which indicated impaired insulin sensitivity.ConclusionsObtained data suggest that ASCs derived from horses older than 5 years old exhibited several molecular alternations which markedly limit their regenerative capacity. The results provide valuable information that allows for a better understanding of the molecular events occurring in ASCs in the course of aging and may help to identify new potential drug targets to restore their regenerative potential.Graphical abstract

Sulforaphane-induced epigenetic regulation of Nrf2 expression by DNA methyltransferase in human Caco-2 cells.

The present study aimed to investigate the mechanism underlying sulforaphane-mediated epigenetic regulation of nuclear factor-erythroid derived 2-like 2 (Nrf2) expression in human colon cancer. Proteins were extracted from normal Caco-2 cells using sulforaphane and 5-aza-2'-deoxycytidine (5-Aza) combined with trichostatin A (TSA). The mRNA and protein expression levels and activity of DNA methyltransferase 1 (DNMT1) were determined. Methylation-specific polymerase chain reaction and bisulfite genomic sequencing were also used to measure the methylation levels of CpG sites in the Nrf2 promoter region. Nrf2 expression was measured using reverse transcription-quantitative PCR and western blot analysis. The results demonstrated that sulforaphane did not affect DNMT1 mRNA expression levels. DNMT1 protein expression was inhibited by sulforaphane and 5-Aza co-treatment with TSA. Nrf2 promoter methylation decreased significantly in the sulforaphane group compared with the control group. Nrf2 promoter methylation level in the 5-Aza+TSA group was the lowest among all groups. Nrf2 mRNA levels exhibited significant differences between the sulforaphane-treated and control groups, as well as between the 5-Aza+TSA and control groups, and the sulforaphane-treated and 5-Aza+TSA groups. Nrf2 protein expression was also inhibited by sulforaphane, as well as 5-Aza co-treatment with TSA. The results revealed that sulforaphane may promote demethylation of the Nrf2 promoter region to increase activation of Nrf2, which induces chemoprevention of colon cancer.

Anthocyanin Delphinidin Prevents Neoplastic Transformation of Mouse Skin JB6 P+ Cells: Epigenetic Re-activation of Nrf2-ARE Pathway

Redox imbalance is a major contributor to the pathogenesis of melanoma and nonmelanoma skin cancer. Activation of the nuclear factor E2-related factor 2 (Nrf2) antioxidant responsive element (ARE) pathway is an intrinsic defense mechanism against oxidative stress. Flavonoids such as anthocyanidins, which are found abundantly in fruits and vegetables, have been shown to activate Nrf2. However, the epigenetic and genetic mechanisms by which anthocyanidins modulate the Nrf2-ARE pathway remain poorly understood in the context of skin cancer. In this study, delphinidin, one of the most potent and abundant anthocyanidins in berries, significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic cell transformation in mouse epidermal JB6 P+ cells by 69.4 to 99.4%. The mechanism was elucidated based on observations of increased ARE-driven luciferase activity and elevated mRNA and protein expression of Nrf2 downstream genes, such as heme oxygenase-1 (Ho-1), in JB6 P+ cells. Activation of the Nrf2-ARE pathway was correlated with demethylation of 15 CpG sites in the mouse Nrf2 promoter region between nt - 1226 and - 863 from the transcription start site. The reduced CpG methylation ratio in the Nrf2 promoter region was consistent with observed decreases in the protein expression of DNA methyltransferases 1 (DNMT1), DNMT3a, and class I/II histone deacetylases (HDACs). Overall, our results suggest that delphinidin, an epigenetic demethylating agent of the Nrf2 promoter, can activate the Nrf2-ARE pathway, which can be applied as a potential skin cancer chemopreventive agent.

Potassium bisperoxo (1,10-phenanthroline) oxovanadate suppresses proliferation of hippocampal neuronal cell lines by increasing DNA methyltransferases.

Bisperoxo (1,10-phenanthroline) oxovanadate (BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases (DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells (HT22) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT22 cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT22 cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT22 cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.

Involvement of nerve growth factor in mouse hippocampal neuronal cell line (HT22) differentiation and underlying role of DNA methyltransferases

ABSTRACTDNA methylation is an epigenetic event involved in regulation of gene transcription during cell differentiation. DNA methyltransferases (DNMT) play a role in differentiation of neural stem cells into neurons. The aim of this study was to determine whether nerve growth factor (NGF) was involved in differentiation of mouse hippocampal neuronal cell line (HT22) as assessed by IncuCyte. Quantitative PCR and western blot were used to measure gene and protein expression of DNMT as well as the activity of DNMTs. Treatment with NGF was found to upregulate both gene and protein expressions as well as total activity of DNMTs in differentiating HT22 cells. Compared to undifferentiating cells, the percentage of differentiating cells at S phase increased significantly when incubated with NGF. In undifferentiated cells, NGF failed to induce gene and protein expressions and activity of DNMTs. Data demonstrate that differentiation of HT22 cells by exposure to NGF involve the activation of DNMTs pathway.

MiR-139-5p suppresses osteosarcoma cell growth and invasion through regulating DNMT1

Abstract. BackgroundAccumulating evidence has suggested the crucial roles of differentially expressed miRNAs in osteosarcoma progression. MiR-139-5p was decreased in various cancers. However, the role of miR-139-5p in the development of osteosarcoma and the underlying mechanism remain to be addressed. MethodsMiR-139-5p and DNA methyltransferase-1 (DNMT1) mRNA expressions in osteosarcoma tissues and cells were detected by qRT-PCR and western blot analysis. The effects of miR-139-5p and DNMT1 on osteosarcoma cell migration, invasion and epithelial-mesenchymal transition (EMT) were investigated through cell migration and invasion assays, and western blot analysis. The relationship between miR-139-5p and DNMT1was explored using luciferase reporter analysis and western blot. A xenograft tumor model was employed to verify the effects of miR-139-5p on osteosarcoma. ResultsWe found that miR-139-5p was strikingly decreased in osteosarcoma tissues and cell lines. MiR-139-5p over-expression suppressed osteosarcoma cell growth, migration and invasion, while loss of miR-139-5p promoted osteosarcoma cell proliferation, migration and invasion. Following, we characterized that DNMT1 was a direct target of miR-139-5p that interacted with the 3′-untranslated region of DNMT1. MiR-139-5p regulated a down-regulation in DNMT1 protein expression levels. We also found that DNMT1 expression was increased and negatively correlated with miR-139-5p expression in osteosarcoma tissues clinically. Xenograft tumor analysis suggested that miR-139-5p over-expression reduced tumor growth in osteosarcoma in vivo through decreasing DNMT1 expressions. ConclusionMiR-139-5p suppressed the osteosarcoma progression by reducing DNMT1, supplying new insight into the molecular mechanism uncovering osteosarcoma growth.

The triterpenoid corosolic acid blocks transformation and epigenetically reactivates Nrf2 in TRAMP-C1 prostate cells.

Corosolic acid (CRA) is found in various plants and has been used as a health food supplement worldwide. Although it has been reported that CRA exhibits significant anticancer activity, the effect of this compound on prostate cancer remains unknown. In this study, we investigated the effect of CRA on cellular transformation and the reactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) through epigenetic regulation in TRAMP-C1 prostate cells. Specifically, we found that CRA inhibited anchorage-independent growth of prostate cancer TRAMP-C1 cells but not Nrf2 knockout prostate cancer TRAMP-C1 cells. Moreover, CRA induced mRNA and protein expression of Nrf2, heme oxygenase-1 (HO-1) and NAD(P)H Quinone Oxidoreductase 1 (NQO1). Bisulfite genomic sequencing and methylated DNA immunoprecipitation results revealed that CRA treatment decreased the level of methylation of the first five CpG sites of the Nrf2 promoter. Histone modification was analyzed using a chromatin immunoprecipitation (ChIP) assay, which revealed that CRA treatment increased the acetylation of histone H3 lysine 27 (H3K27ac) while decreasing the trimethylation of histone H3 lysine 27 (H3K27me3) in the promoter region of Nrf2. Furthermore, CRA treatment attenuated the protein expression of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). These findings indicate that CRA has a significant anticancer effect in TRAMP-C1 cells, which could be partly attributed to epigenetics including its ability to epigenetically restore the expression of Nrf2.

HOTAIR-mediated reciprocal regulation of EZH2 and DNMT1 contribute to polyphyllin I-inhibited growth of castration-resistant prostate cancer cells in vitro and in vivo

BackgroundPolyphyllin I (PPI), one of the steroidal saponins in paris polyphylla, has been reported to exhibit antitumor effects. However, the detailed molecular mechanism underlying this has not been elucidated. MethodsCell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Flow cytometry assays, respectively. Cell invasion and migration were examined by Transwell invasion and wound healing assays. Western blot analysis was performed to examine the protein expressions of zeste homolog 2 (EZH2), DNA methyltransferase 1 (DNMT1). QRT-PCR was used to examine the levels of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR). Small interfering RNAs (siRNAs) method was used to knockdown HOTAIR. Exogenously expressions of HOTAIR, DNMT1 and EZH2 were carried out by Transient transfection assays. EZH2 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. A nude mice xenograft model was used to confirm the findings in vitro. ResultsWe showed that PPI significantly inhibited growth, induced cell cycle arrest of castration-resistant prostate cancer (CRPC) cells. In addition, PPI also reduced the migration and invasion in CRPC cells. In mechanism, we found that PPI decreased the protein expressions of EZH2, DNMT1 and levels of HOTAIR. Interestingly, silenced HOTAIR reduced EZH2 and DNMT1 protein expressions. On the contrary, exogenously expressed HOTAIR resisted PPI-inhibited EZH2 and DNMT1 protein expressions, EZH2 promoter activity and cell growth. Moreover, excessive EZH2 antagonized PPI-suppressed DNMT1 protein expression or vice versa. Consistent with this, PPI inhibited tumor growth, HOTAIR, the protein expressions of DNMT1 and EZH2 in vivo. ConclusionOur results show that PPI inhibits growth of CRPC cells through inhibition of HOTAIR expression, subsequently; this results in the repression of DNMT1 and EZH2 expressions. The interactions among HOTAIR, DNMT1 and EZH2, and reciprocal regulation of DNMT1 and EZH2 contribute to the overall responses of PPI. This study reveals a novel mechanism for HOTAIR-mediated regulating DNMT1 and EZH2 in response to PPI in inhibition of the growth of CRPC cells.