Smoking changes adaptive immunity with persistent effects.

Individuals differ widely in their immune responses, with age, sex and genetic factors having major roles in this inherent variability1-6. However, the variables that drive such differences in cytokine secretion-a crucial component of the host response to immune challenges-remain poorly defined. Here we investigated 136 variables and identified smoking, cytomegalovirus latent infection and body mass index as major contributors to variability in cytokine response, with effects of comparable magnitudes with age, sex and genetics. We find that smoking influences both innate and adaptive immune responses. Notably, its effect on innate responses is quickly lost after smoking cessation and is specifically associated with plasma levels of CEACAM6, whereas its effect on adaptive responses persists long after individuals quit smoking and is associated with epigenetic memory. This is supported by the association of the past smoking effect on cytokine responses with DNA methylation at specific signal trans-activators and regulators of metabolism. Our findings identify three novel variables associated with cytokine secretion variability and reveal roles for smoking in the short- and long-term regulation of immune responses. These results have potential clinical implications for the risk of developing infections, cancers or autoimmune diseases.

Pharmacological inhibition of DNMT1 restores macrophage autophagy and M2 polarization in Western diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is associated with an increased ratio of classically activated M1 macrophages/Kupffer cells to alternatively activated M2 macrophages, which plays an imperative role in the development and progression of NAFLD. However, little is known about the precise mechanism behind macrophage polarization shift. Here, we provide evidence regarding the relationship between the polarization shift in Kupffer cells and autophagy resulting from lipid exposure. High-fat and high-fructose diet supplementation for 10 weeks significantly increased the abundance of Kupffer cells with an M1-predominant phenotype in mice. Interestingly, at the molecular level, we also observed a concomitant increase in expression of DNA methyltransferases DNMT1 and reduced autophagy in the NAFLD mice. We also observed hypermethylation at the promotor regions of autophagy genes (LC3B, ATG-5, and ATG-7). Furthermore, the pharmacological inhibition of DNMT1 by using DNA hypomethylating agents (azacitidine and zebularine) restored Kupffer cell autophagy, M1/M2 polarization, and therefore prevented the progression of NAFLD. We report the presence of a link between epigenetic regulation of autophagy gene and macrophage polarization switch. We provide the evidence that epigenetic modulators restore the lipid-induced imbalance in macrophage polarization, therefore preventing the development and progression of NAFLD.

The Immuno-Oncology and Genomic Aspects of DNA-Hypomethylating Therapeutics in Acute Myeloid Leukemia.

Hypomethylating agents (HMAs) have been used for decades in the treatment of hematologic neoplasms, and now, have gathered attention again in terms of their combination with potent molecular-targeted agents such as a BCL-6 inhibitor venetoclax and an IDH1 inhibitor ivosidenib, as well as a novel immune-checkpoint inhibitor (anit-CD47 antibody) megrolimab. Several studies have shown that leukemic cells have a distinct immunological microenvironment, which is at least partially due to genetic alterations such as the TP53 mutation and epigenetic dysregulation. HMAs possibly improve intrinsic anti-leukemic immunity and sensitivity to immune therapies such as PD-1/PD-L1 inhibitors and anti-CD47 agents. This review describes the immuno-oncological backgrounds of the leukemic microenvironment and the therapeutic mechanisms of HMAs, as well as current clinical trials of HMAs and/or venetoclax-based combination therapies.

Influence of antidepressant treatment on SLC6A4 methylation in Korean patients with major depression.

Genetic variation of the serotonin transporter gene (SLC6A4) has been suggested as potential mediator for antidepressant response in patients with depression. This study aimed to determine whether DNA methylation in SLC6A4 changes after antidepressant treatment and whether it affects treatment response in patients with depression. Overall, 221 Korean patients with depression completed 6 weeks of selective serotonin reuptake inhibitor (SSRI) monotherapy. DNA was extracted from venous blood pre- and post-treatment, and DNA methylation was analyzed using polymerase chain reaction. We used Wilcoxon's signed-rank test to verify the difference in methylation after treatment. Treatment response was assessed using the 17-item Hamilton Depression Rating Scale, and mRNA levels were quantified. After adjusting for relevant covariates, DNA methylation was significantly altered in specific CpG sites in SLC6A4 (p < .001 in CpG3, CpG4, and CpG5) following 6 weeks of treatment. Methylation change's magnitude (ΔDNA methylation) after drug treatment was not associated with treatment response or mRNA level change. SSRI antidepressants can influence SLC6A4 methylation in patients with depression. However, ΔDNA methylation at CpG3, CpG4, and CpG5 in SLC6A4 was not associated with treatment response. Future studies should investigate the integrative effect of other genetic variants and CpG methylation on gene transcription and antidepressant treatment response.

DNA methylation 6 mA and histone methylation involved in multi-/trans-generational reproductive effects in Caenorhabditis elegans induced by Atrazine.

Atrazine (ATR), a widely used triazine herbicide, is an environmental endocrine disruptor that can cause health problems. However, whether there are multi/trans-generational reproductive impacts of ATR have not been studied. Therefore, in this study, Caenorhabditis elegans was used as a preferable model organism to identify the multi/trans-generational reproductive toxicity of ATR. Only parental C.elegans (P0) were exposed to different concentrations (0.0004-40 mg/L) for 48 h and the subsequent offspring (F1-F5) were grown under ATR-free conditions and ATR conditions.The results showed that ATR exposure during P0 decreased fecundity, including a reduction in fertilized eggs, oocytes, and ovulation rate, delayed gonadal development, and decreased the relative area of gonad arm and germ cell number. Furthermore, continuous ATR exposure (P0-F5) causes a significant increase in reproductive toxicity in subsequent generations, although no significant toxicity occurred in the P0 generation after exposure to environmental-related concentrations, suggesting that ATR exposure might have cumulative effects. Likewise, parental exposure to ATR caused transgenerational toxicity impairments. Interestingly, only reproductive toxicity, not development toxicity, was transmitted to several generations (F1-F4), and the F2 generation showed the most notable changes. QRT-PCR results showed that genes expression related to DNA methylation 6 mA (damt-1, nmad-1) and histone H3 methylation (mes-4, met-2, set-25, set-2, and utx-1) can also be passed on to offspring. The function of H3K4 and H3K9 methylation were explored by using loss-of-function mutants for set-2, set-25, and met-2. Transmissible reproductive toxicity was absent in met-2(n4256), set-2(ok952), and set-25(n5021) mutants, which suggests that the histone methyltransferases H3K4 and H3K9 activity are indispensable for the transgenerational effect of ATR. Finally, the downstream genes of DNA methylation and histone H3 methylation were determined. ATR upregulated the expression of ZC317.7, hsp-6, and hsp-60. Mitochondrial stress in parental generation dependent transcription 6 mA modifiers may establish these epigenetic marks in progeny.

The impact of pre-pregnancy folic acid intake on placental DNA methylation in a fortified cohort.

Folate plays an important role in the modulation of one-carbon metabolism and DNA methylation through a complex biosynthesis pathway. Folate deficiency during pregnancy has been associated with an increased risk for birth defects. This study investigates the extent to which the availability of folate and S-Adenosylmethionine (SAM) affects placental DNA methylation. We hypothesized that maintaining sufficient levels of folate and SAM is particularly important in individuals carrying the MTHFR C677T polymorphism. Maternal- and cord blood was analyzed to genotype the MTHFR rs1801133 SNP. Red blood cell (RBC) folate, vitamin B12, SAM, and S-Adenosylhomocysteine (SAH) were analyzed in cord blood. Epigenome-wide methylation analyses were performed on 90 placenta tissue samples isolated from the fetal side of the placenta; 45 originating from mother-infant dyads homozygous for the MTHFR C677T variant and 45 originating from mother-infant dyads with the homozygous wild type MTHFR677 genotype. Verification of the results was performed using pyrosequencing assays. Genome-wide placental DNA methylation patterns were relatively stable and not significantly affected by levels of one-carbon metabolites. MTHFR genotype was associated with DNA methylation of several loci, including a locus in the MTHFR region. RBC folate and particularly the SAM:SAH ratio did affect overall CpG DNA methylation in some CpG regions when the loci were split according to their CpG island relation. This was most evident in participants carrying the MTHFR C677T variant suggesting a stronger influence of the biosynthesis pathway on the overall placental DNA methylation in MTHFR TT individuals than in MTHFR CC individuals.

Cromolyn chitosan nanoparticles reverse the DNA methylation of RASSF1A and p16 genes and mitigate DNMT1 and METTL3 expression in breast cancer cell line and tumor xenograft model in mice.

BACKGROUND: Developing epigenetic drugs for breast cancer (BC) remains a novel therapeutic approach. Cromolyn is a mast cell stabilizer emerging as an anticancer drug; its encapsulation in chitosan nanoparticles (CSNPs) improves its effect and bioavailability. However, its effect on DNA and RNA methylation machineries has not been previously tackled. METHODS: The possible anticancer effect of cromolyn CSNPs and its potential as an epigenetic drug was investigated in vitro using MCF-7 human BC cell line and in vivo using Ehrlich ascites carcinoma-xenograft model in mice symbolizing murine mammary adenocarcinoma. Mice were injected with a single dose of Ehrlich ascites carcinoma cells subcutaneously for the induction of tumor mass, and then randomized into three groups: control, cromolyn CSNPs (equivalent to 5 mg cromolyn/kg, i.p.) and plain CSNPs twice/week for 2 weeks. RESULTS: Cromolyn CSNPs showed prominent anticancer effect in MCF-7 cells by reducing the cell viability percent and enhancing DNA damage in the comet assay demonstrating its apoptotic actions. Mechanistically, cromolyn CSNPs influenced potential epigenetic processes through mitigating DNA methyltransferase 1 (DNMT1) expression, reversing the hypermethylation pattern of the tumor suppressor RASSF1A and p16 genes and attenuating the expression of the RNA N6-methyladenosine writer, methyltransferase-like 3 (METTL3). Cromolyn CSNPs diminished ERK1/2 phosphorylation, a possible arm influencing DNMT1 expression. In vivo, cromolyn CSNPs lessened the tumor volume and halted DNMT1 and METTL3 expression in Ehrlich carcinoma mice. CONCLUSIONS: Cromolyn CSNPs have the premise as an epigenetic drug through inhibiting ERK1/2 phosphorylation/DNMT1/DNA methylation and possibly impacting the RNA methylation machinery via mitigating METTL3 expression.

Genome-wide DNA methylation signature predict clinical benefit of bevacizumab in non-small cell lung cancer.

BACKGROUND: The efficacy of bevacizumab in non-small cell lung cancer (NSCLC) patients is unsatisfactory, and the selection of suitable patients is still challenging. Given the epigenetic modifications can contribute to an aberrant regulation of angiogenesis and microenvironment, we investigated DNA methylation profiles to determine clinical benefit of bevacizumab in NSCLC patients. METHODS: Genome-wide DNA methylation profiling was performed in NSCLC patients treated with chemotherapy in combination with bevacizumab. Patients were divided into better prognosis group (A group) and inferior prognosis group (B group) based on their survival. The difference of methylation patterns and respective functional enrichment analysis were performed between two groups. Prognostic DNA methylation signature for bevacizumab was established with the least absolute shrinkage and selection operator regression analyses. TISIDB database was further used to infer immunological relationship for prognostic related DNA methylation. RESULTS: Twenty patients were included in this study, and significantly distinct methylation patterns were observed between patients with different prognosis. Related genes of different methylation regions were significantly enriched in the biological process of cell projection assembly, neutrophil mediated immunity, and pathway of VEGFA-VEGFR2 signaling pathway, neutrophil degranulation. A 10-gene DNA methylation signature for prognosis prediction was established with the C-index of 0.76. And host genes of signature were found to be related to the abundance of ActCD4, Th1, ActCD8, NKT and neutrophil cells. CONCLUSION: The 10-gene DNA methylation signature could serve as a novel biomarker to predict the clinical benefit of bevacizumab therapy and improve this anti-tumor approach for NSCLC patients.

Ibuprofen promotes p75 neurotrophin receptor expression through modifying promoter methylation and N6-methyladenosine-RNA-methylation in human gastric cancer cells.

It is acknowledged that nonsteroidal anti-inflammatory drugs (NSAIDs) can participate in various signaling pathways, while information about their epigenetic effects are limited. p75NTR (p75 neurotrophin receptor) can inhibit tumor growth by inducing cell cycle arrest and regulating cell cycle arrest and apoptotic cell death. The expression of p75NTR is influenced by epigenetic roles. We explored the effects of ibuprofen on p75NTR expression and investigated whether promoter methylation and N6-methyladenosine (m6A) RNA methylation regulates this process in human gastric cancer cells (SGC7901 and MKN45). Cell lines were treated with ibuprofen 0, 2.5, 5, 10, 20 µM, and then DNA, RNA, and protein were isolated 24 h later. Expression and promoter methylation of p75NTR were detected by RT-qPCR and Western blot. The levels of m6A-p75NTR were measured by RNA immunoprecipitation. We also used RT-qPCR to determine the levels of m6A-related regulators, METTL3, METTL14, ALKBH5, FTO, YTHDC2, and YTHDF1-3. Ibuprofen attenuated p75NTR promoter methylation (p < 0.01) and increased p75NTR level (p < 0.001). Ibuprofen increased m6A-p53 expression (p < 0.01) by promoting the expression of METTL3 (p < 0.01) and METTL14 (p < 0.05); and increased levels of YTHDF1 (p < 0.001), YTHDF3 (p < 0.001), and YTHDC2 (p < 0.01) that finally reinforced p53 translation (p < 0.01). Therefore, our results present that ibuprofen epigenetically increased p75NTR expression by downregulating promoter methylation and upregulating m6A-RNA-methylation in SGC7901 and MKN45 cells. Our study unveils a novel mechanism for p75NTR regulation by NSAIDs and helps the design of treatment targets.

Cocaine-Induced Changes in Sperm Cdkn1a Methylation Are Associated with Cocaine Resistance in Male Offspring.

Paternal environmental perturbations can influence the physiology and behavior of offspring. For example, our previous work showed reduced cocaine reinforcement in male, but not female, progeny of rat sires that self-administered cocaine. The information transfer from sire to progeny may occur through epigenetic marks in sperm, encompassing alterations in small noncoding RNAs, including microRNAs (miRNAs) and/or DNA methylation. Here, no reliable changes in miRNAs in the sperm of cocaine- relative to saline-experienced sires were identified. In contrast, 272 differentially methylated regions were observed in sperm between these groups. Two hypomethylated promoter regions in the sperm of cocaine-experienced rats were upstream of cyclin-dependent kinase inhibitor 1a (Cdkn1a). Cdkn1a mRNA also was selectively increased in the NAc of cocaine-sired male (but not female) offspring. Cocaine self-administration also enhanced Cdkn1a expression in the accumbens of cocaine-sired rats. These results suggest that changes in Cdkn1a may play a role in the reduced cocaine reinforcing efficacy observed in cocaine-sired male rats. Introducing a 90 d delay between sire self-administration and breeding reversed both cocaine resistance and the increase in accumbens Cdkn1a mRNA in male offspring, indicating that cocaine-induced epigenetic modifications are eliminated with sperm turnover. Collectively, our results indicate that cocaine self-administration produces hypomethylation of Cdkn1a in sperm and a selective increase in the expression of this gene in the NAc of male offspring, which is associated with blunted cocaine reinforcement.SIGNIFICANCE STATEMENT The relatively new field of transgenerational epigenetics explores the effects of environmental perturbations on offspring behavior and physiology. Our prior work in rats indicated that male, but not female, progeny of sires that self-administered cocaine displayed reduced cocaine reinforcement. The information transfer from sire to progeny may occur through heritable epigenetic marks in sperm, including DNA methylation. The present findings revealed two hypomethylated promoter regions upstream of the Cdkn1a gene in sire sperm. Remarkably, Cdkn1a expression was selectively decreased in offspring NAc, a brain region that regulates cocaine reinforcement.

Genome-Wide DNA Methylation in Policemen Working in Cities Differing by Major Sources of Air Pollution.

DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and it can serve as a useful biomarker of prior environmental exposure and future health outcomes. This study focused on DNA methylation profiles in a human cohort, comprising 125 nonsmoking city policemen (sampled twice), living and working in three localities (Prague, Ostrava and Ceske Budejovice) of the Czech Republic, who spent the majority of their working time outdoors. The main characterization of the localities, differing by major sources of air pollution, was defined by the stationary air pollution monitoring of PM2.5, B[a]P and NO2. DNA methylation was analyzed by a genome-wide microarray method. No season-specific DNA methylation pattern was discovered; however, we identified 13,643 differentially methylated CpG loci (DML) for a comparison between the Prague and Ostrava groups. The most significant DML was cg10123377 (log2FC = -1.92, p = 8.30 × 10-4) and loci annotated to RPTOR (total 20 CpG loci). We also found two hypomethylated loci annotated to the DNA repair gene XRCC5. Groups of DML annotated to the same gene were linked to diabetes mellitus (KCNQ1), respiratory diseases (PTPRN2), the dopaminergic system of the brain and neurodegenerative diseases (NR4A2). The most significant possibly affected pathway was Axon guidance, with 86 potentially deregulated genes near DML. The cluster of gene sets that could be affected by DNA methylation in the Ostrava groups mainly includes the neuronal functions and biological processes of cell junctions and adhesion assembly. The study demonstrates that the differences in the type of air pollution between localities can affect a unique change in DNA methylation profiles across the human genome.

Subchronic PCP effects on DNA methylation and protein expression of NMDA receptor subunit genes in the prefrontal cortex and hippocampus of female rats.

BACKGROUND: N-methyl-d-aspartate receptor (NMDAR) dysfunction is implicated in schizophrenia, and NMDAR antagonists, such as phencyclidine (PCP), can induce behaviours that mimic aspects of the disorder. AIMS: We investigated DNA methylation of Grin1, Grin2a and Grin2b promoter region and NR1 and NR2 protein expression in the prefrontal cortex (PFC) and hippocampus of adult female Lister-hooded rats following subchronic PCP (scPCP) administration. We also determined whether any alterations were tissue-specific. METHODS: Rats were divided into two groups that received vehicle (0.9% saline) or 2 mg/kg PCP twice a day for 7 days (n = 10 per group). After behavioural testing (novel object recognition), to confirm a cognitive deficit, brains were dissected and NMDAR subunit DNA methylation and protein expression were analysed by pyrosequencing and ELISA. Line-1 methylation was determined as a measure of global methylation. Data were analysed using Student's t-test and Pearson correlation. RESULTS: The scPCP administration led to Grin1 and Grin2b hypermethylation and reduction in NR1 protein in both PFC and hippocampus. No significant differences were observed in Line-1 or Grin2a methylation and NR2 protein. CONCLUSIONS: The scPCP treatment resulted in increased DNA methylation at promoter sites of Grin1 and Grin2b NMDAR subunits in two brain areas implicated in schizophrenia, independent of any global change in DNA methylation, and are similar to our observations in a neurodevelopmental animal model of schizophrenia - social isolation rearing post-weaning. Moreover, these alterations may contribute to the changes in protein expression for NMDAR subunits demonstrating the potential importance of epigenetic mechanisms in schizophrenia.

Ferroptosis inhibitor ferrostatin­1 alleviates homocysteine­induced ovarian granulosa cell injury by regulating TET activity and DNA methylation.

Polycystic ovary syndrome is one of the most common endocrine and metabolic gynecological disorders, of which dysfunction of ovarian granulosa cells is a key contributing factor. The aim of the present study was to explore the role of ferrostatin­1 (Fer­1), a ferroptosis inhibitor, in a cell injury model established by homocysteine (Hcy)­induced ovarian granulosa KGN cell line and the potential underlying mechanism. Cell viability was measured using Cell Counting Kit­8 assay in the presence or absence of Hcy and Fer­1. Cell apoptosis was assessed using TUNEL staining and the expression levels of apoptosis­related proteins were measured using western blotting. To explore the effects of Fer­1 on oxidative stress in Hcy­treated ovarian granulosa cells, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), lactate dehydrogenase (LDH) and glutathione (GSH) were measured using their corresponding kits. Furthermore, Fe2+ levels were assessed using Phen Green™ SK labeling and western blotting was performed to measure the protein expression levels of ferroptosis­associated proteins GPX4, SLC7A11, ASCL4 and DMT1. Subsequently, DNA methylation and ten­eleven translocation (TET) 1/2 demethylase levels were also detected to evaluate the extent of overall DNA methylation in ovarian granulosa cells after Hcy treatment. The TET1/2 inhibitor Bobcat339 hydrochloride was applied to treat ovarian granulosa cells before evaluating the possible effects of Fer­1 on TET1/2 and DNA methylation. Fer­1 was found to markedly elevate ovarian granulosa cell viability following Hcy treatment. The apoptosis rate in Fer­1­treated groups was also markedly decreased, which was accompanied by downregulated Bax and cleaved caspase­3 expression and upregulated Bcl­2 protein expression. In addition, Fer­1 treatment reduced the levels of ROS, MDA and LDH whilst enhancing the levels of GSH. Fe2+ levels were significantly decreased following Fer­1 treatment, which also elevated glutathione peroxidase 4 expression whilst reducing solute carrier family 7 member 11, achaete­scute family BHLH transcription factor 4 and divalent metal transporter 1 protein expression. Fer­1 significantly inhibited DNA methylation and enhanced TET1/2 levels, which were reversed by treatment with Bobcat339 hydrochloride. Subsequent experiments on cell viability, oxidative stress, Fe2+ content, ferroptosis­ and apoptosis­related proteins levels revealed that Bobcat339 hydrochloride reversed the effects of Fer­1 on ovarian granulosa Hcy­induced cell injury. These results suggest that Fer­1 may potentially protect ovarian granulosa cells against Hcy­induced injury by increasing TET levels and reducing DNA methylation.

Methylation­associated inactivation of JPH3 and its effect on prognosis and cell biological function in HCC.

In recent years, researchers have found that epigenetics plays an important role in the occurrence and development of hepatocellular carcinoma (HCC). DNA methylation is involved in the proliferation and metastasis of HCC. However, the junctophilin 3 (JPH3) level and the potential regulatory mechanism of its DNA methylation in HCC remain uncertain. In the present study, 73 HCC samples were enrolled to analyze the expression of JPH3. Reverse­transcription quantitative PCR, western blotting and immunohistochemistry were used to detect the expression of JPH3 in HCC. Kaplan­Meier method and Cox regression analysis were applied to evaluate the prognostic impact of JPH3 on HCC patients. DNA methylation­specific PCR and bisulfite Sanger sequencing were used to detect the degree of DNA methylation of JPH3 in HCC. The demethylation drug 5­Aza­2'­deoxycytidine (5­Aza) was used to reduce the DNA methylation of JPH3. The role of JPH3 in the malignant biological behavior of HCC by promoting epithelial­mesenchymal transition (EMT) was confirmed by functional cell experiments. The results showed that JPH3 exhibited low levels in HCC tissues and cell lines. HCC patients with low expression of JPH3 had poor survival outcomes. JPH3 had higher DNA methylation levels in HCC tissues and cell lines. When the demethylation drug 5­Aza was used to reduce the degree of methylation of JPH3, its protein expression level was significantly increased and it significantly inhibited the malignant biological behavior of HCC cells. Additionally, effective increase in the expression of JPH3 through gene regulation technology also inhibited the proliferation, invasion and migration of HCC cells. After altering the DNA methylation level of JPH3, the EMT of HCC cells was also affected. Therefore, our study demonstrated the inactivation of JPH3 by promoter methylation and its function as a tumor suppressor in HCC. JPH3 may serve as a biomarker for early diagnosis and as a potential therapeutic target for HCC.

A Mixture of Endocrine Disrupting Chemicals Associated with Lower Birth Weight in Children Induces Adipogenesis and DNA Methylation Changes in Human Mesenchymal Stem Cells.

Endocrine Disrupting Chemicals (EDCs) are man-made compounds that alter functions of the endocrine system. Environmental mixtures of EDCs might have adverse effects on human health, even though their individual concentrations are below regulatory levels of concerns. However, studies identifying and experimentally testing adverse effects of real-life mixtures are scarce. In this study, we aimed at evaluating an epidemiologically identified EDC mixture in an experimental setting to delineate its cellular and epigenetic effects. The mixture was established using data from the Swedish Environmental Longitudinal Mother and child Asthma and allergy (SELMA) study where it was associated with lower birth weight, an early marker for prenatal metabolic programming. This mixture was then tested for its ability to change metabolic programming of human mesenchymal stem cells. In these cells, we assessed if the mixture induced adipogenesis and genome-wide DNA methylation changes. The mixture increased lipid droplet accumulation already at concentrations corresponding to levels measured in the pregnant women of the SELMA study. Furthermore, we identified differentially methylated regions in genes important for adipogenesis and thermogenesis. This study shows that a mixture reflecting human real-life exposure can induce molecular and cellular changes during development that could underlie adverse outcomes.

The effect of benzo[alpha]pyrene on DNA methylation and telomerase activity in human normal and cancer cells.

Benzo[a]pyrene (BaP) exposure has been associated with an increased risk of carcinogenesis. We investigated the effects of BaP on cell viability, the promoter methylation of 11 tumor-associated genes, the global DNA methylation, and telomerase enzyme activity in 5 human cancer cell lines (HCT116, PC3, MDA-MB-231, A549, and HepG2) and normal human peripheral blood mononuclear cells (PBMCs) and adipose-derived mesenchymal stem cells (AD-MSCs). BaP inhibited the proliferation of cells in a dose-dependent manner, as measured by MTT assay. Human normal cells were more sensitive to BaP cytotoxicity than cancer cells. After treatment with the minimally toxic concentration of BaP (5 µM for 72 h), 3 differentially methylated genes (genes with different promoter methylation status) were identified between BaP-treated and untreated control cells, as verified by MSP analysis. BaP induced hypomethylation of COX-2 and MSH2 in normal PBMCs and hypermethylation of APC in HCT116 CRC cells. BaP also non-significantly decreased global methylation levels in 3 cancer cell lines (HCT16, PC3, and A549), as measured by ELISA assay. BaP also reduced telomerase enzyme activity in human AD-MSC cells in a dose-dependent manner. To our knowledge, this is the first report of BaP-effects on telomerase activity and DNA methylation in human normal and cancer cells.

Iodine-125 seed represses the growth and facilitates the apoptosis of colorectal cancer cells by suppressing the methylation of miR-615 promoter.

BACKGROUND: Colorectal cancer (CRC) represents a common malignancy in gastrointestinal tract. Iodine-125 (125I) seed implantation is an emerging treatment technology for unresectable tumors. This study investigated the mechanism of 125I seed in the function of CRC cells. METHODS: The CRC cells were irradiated with different doses of 125I seed (0.4, 0.6 and 0.8 mCi). miR-615 expression in CRC tissues and adjacent tissues was detected by RT-qPCR. miR-615 expression was intervened with miR-615 mimic or miR-615 inhibitor, and then the CRC cells were treated with 5-AZA (methylation inhibitor). The CRC cell growth, invasion and apoptosis were measured. The methylation level of miR-615 promoter region was detected. The xenograft tumor model irradiated by 125I seed was established in nude mice. The methylation of miR-615, Ki67 expression and CRC cell apoptosis were detected. RESULTS: 125I seed irradiation repressed the growth and facilitated apoptosis of CRC cells in a dose-dependent manner. Compared with adjacent tissues, miR-615 expression in CRC tissues was downregulated and miR-615 was poorly expressed in CRC cells. Overexpression of miR-615 suppressed the growth of CRC cells. 125I seed-irradiated CRC cells showed increased miR-615 expression, reduced growth rate and enhanced apoptosis. The methylation level of miR-615 promoter region in CRC cells was decreased after 125I seed treatment. In vivo experiments confirmed that 125I seed-irradiated xenograft tumors showed reduced methylation of the miR-615 promoter and increased miR-615 expression, as well as decreased Ki67 expression and enhanced apoptosis. The target genes of miR-615 and its regulatory downstream pathway were further predicted by bioinformatics analysis. CONCLUSIONS: 125I seed repressed the growth and facilitated the apoptosis of CRC cells by suppressing the methylation of the miR-615 promoter and thus activating miR-615 expression. The possible mechanism was that miR-615-5p targeted MAPK13, thus affecting the MAPK pathway and the progression of CRC.

HDAC8 suppresses the epithelial phenotype and promotes EMT in chemotherapy-treated basal-like breast cancer.

BACKGROUND: Basal-like breast cancer (BLBC) is one of the most aggressive malignant diseases in women with an increased metastatic behavior and poor prognosis compared to other molecular subtypes of breast cancer. Resistance to chemotherapy is the main cause of treatment failure in BLBC. Therefore, novel therapeutic strategies counteracting the gain of aggressiveness underlying therapy resistance are urgently needed. The epithelial-to-mesenchymal transition (EMT) has been established as one central process stimulating cancer cell migratory capacity but also acquisition of chemotherapy-resistant properties. In this study, we aimed to uncover epigenetic factors involved in the EMT-transcriptional program occurring in BLBC cells surviving conventional chemotherapy. RESULTS: Using whole transcriptome data from a murine mammary carcinoma cell line (pG-2), we identified upregulation of Hdac4, 7 and 8 in tumor cells surviving conventional chemotherapy. Subsequent analyses of human BLBC patient datasets and cell lines established HDAC8 as the most promising factor sustaining tumor cell viability. ChIP-sequencing data analysis identified a pronounced loss of H3K27ac at regulatory regions of master transcription factors (TFs) of epithelial phenotype like Gata3, Elf5, Rora and Grhl2 upon chemotherapy. Interestingly, impairment of HDAC8 activity reverted epithelial-TFs levels. Furthermore, loss of HDAC8 activity sensitized tumor cells to chemotherapeutic treatments, even at low doses. CONCLUSION: The current study reveals a previously unknown transcriptional repressive function of HDAC8 exerted on a panel of transcription factors involved in the maintenance of epithelial cell phenotype, thereby supporting BLBC cell survival to conventional chemotherapy. Our data establish HDAC8 as an attractive therapeutically targetable epigenetic factor to increase the efficiency of chemotherapeutics.

The tyrosine kinase inhibitor LPM4870108 impairs learning and memory and induces transcriptomic and gene­specific DNA methylation changes in rats.

Tyrosine kinase inhibitors (TKIs), which have been developed and approved for cancer treatment in the last few years, are involved in synaptic plasticity of learning and memory. Epigenetic modifications also play crucial roles in the process of learning and memory, but its relationship with TKI-induced learning and memory impairment has not been investigated. We hypothesized that LPM4870108, an effective anti-cancer Trk inhibitor, might affect the learning and memory via epigenetic modifications. In this study, rats were orally administered with LPM4870108 (0, 1.25, 2.5, or 5.0 mg/kg) twice daily for 28 days, after which animals were subjected to a Morris water maze test. LPM4870108 exposure caused learning and memory impairments in this test in a dose-dependent manner and reduced the spine densities. Whole-genome transcriptomic analysis revealed significant differences in the patterns of hippocampal gene expression in LPM4870108-treated rats. These transcriptomic data were combined with next-generation bisulfite sequencing analysis, after which RT-PCR and pyrosequencing were conducted, revealing epigenetic alterations associated with genes (Snx8, Fgfr1, Dusp4, Vav2, and Satb2) known to regulate learning and memory. Increased mRNA and protein expression levels of hippocampal Dnmt1 and Dnmt3a were also observed in these rats. Overall, these data suggest that gene-specific alterations in patterns of DNA methylation can potentially contribute to the incidence of learning and memory deficits associated with exposure to LPM4870108.