4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced Histone Acetylation via α7nAChR-Mediated PI3K/Akt Activation and Its Impact on γ-H2AX Generation.

A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

Involvement of Neutrophil Extracellular Traps in Cerebral Arteriovenous Malformations.

BACKGROUND: Cerebral arteriovenous malformations (cAVMs) represent tangles of abnormal vasculature without intervening capillaries. High-pressure vascular channels due to abnormal arterial and venous shunts can lead to rupture. Multiple pathways are involved in the pathobiology of cAVMs including inflammation and genetic factors such as KRAS mutations. Neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs), plays a multifunctional role in infection, inflammation, thrombosis, intracranial aneurysms, and tumor progression. However, the relationship between NETs and the pathobiology of cAVMs remains unknown. We tested whether NETs play a role in the pathobiology of cAVMs. METHODS: We analyzed samples from patients who had undergone surgery for cAVM and immunohistochemically investigated expression of citrullinated histone H3 (CitH3) as a marker of NETs. CitH3 expression was compared among samples from cAVM patients, epilepsy patients, and normal human brain tissue. Expressions of thrombotic and inflammatory markers were also examined immunohistochemically in samples from cAVM patients. RESULTS: Expression of CitH3 derived from neutrophils was observed intravascularly in all cAVM samples but not other samples. Nidi of AVMs showed migration of many Iba-I-positive cells adjacent to the endothelium and endothelial COX2 expression, accompanied by expression of IL-6 and IL-8 in the endothelium and intravascular neutrophils. Unexpectedly, expression of CitH3 was not necessarily localized to the vascular wall and thrombus. CONCLUSIONS: Our results offer the first evidence of intravascular expression of NETs, which might be associated with vascular inflammation in cAVMs.

The COP9 signalosome subunit 3 is necessary for early embryo survival by way of a stable protein deposit in mouse oocytes.

Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as 'Trim away' (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing.

The tumour suppressor brain tumour (Brat) regulates linker histone dBigH1 expression in the Drosophila female germline and the early embryo.

Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. Here, we address this question in Drosophila, which encodes for a single germline-specific dBigH1 linker histone. We show that during female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. We find that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, we discuss the situation in testes, where Brat is not expressed, but dBigH1 is translationally silenced too.

PRSS50 is a testis protease responsible for proper sperm tail formation and function.

Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenoteratozoospermia. We have identified protease serine 50 (PRSS50) as having a crucial role in sperm development, because Prss50-null mice presented with impaired fertility and sperm tail abnormalities. PRSS50 could also be involved in centrosome function because these mice showed a threefold increase in acephalic sperm (head-tail junction defect), sperm with multiple heads (spermatid division defect) and sperm with multiple tails, including novel two conjoined sperm (complete or partial parts of several flagellum on the same plasma membrane). Our data support that, in the testis, as in tumorigenesis, PRSS50 activates NFκB target genes, such as the centromere protein leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1), which is required for heterochromatin maintenance. Prss50-null testes have increased IκκB, and reduced LRWD1 and histone expression. Low levels of de-repressed histone markers, such as H3K9me3, in the Prss50-null mouse testis may cause increases in post-meiosis proteins, such as AKAP4, affecting sperm formation. We provide important insights into the complex mechanisms of sperm development, the importance of testis proteases in fertility and a novel mechanism for MMAF.

Tumor-specific overexpression of histone gene, H3C14 in gastric cancer is mediated through EGFR-FOXC1 axis.

Incorporation of different H3 histone isoforms/variants have been reported to differentially regulate gene expression via alteration in chromatin organization during diverse cellular processes. However, the differential expression of highly conserved histone H3.2 genes, H3C14 and H3C13 in human cancer has not been delineated. In this study, we investigated the expression of H3.2 genes in primary human gastric, brain, breast, colon, liver, and head and neck cancer tissues and tumor cell lines. The data showed overexpression of H3.2 transcripts in tumor samples and cell lines with respect to normal counterparts. Furthermore, TCGA data of individual and TCGA PANCAN cohort also showed significant up-regulation of H3.2 genes. Further, overexpressed H3C14 gene coding for H3.2 protein was regulated by FOXC1 transcription factor and G4-cassette in gastric cancer cell lines. Elevated expression of FOXC1 protein and transcripts were also observed in human gastric cancer samples and cell lines. Further, FOXC1 protein was predominantly localized in the nuclei of neoplastic gastric cells compared to normal counterpart. In continuation, studies with EGF induction, FOXC1 knockdown, and ChIP-qPCR for the first time identified a novel axis, EGFR-FOXC1-H3C14 for regulation of H3C14 gene overexpression in gastric cancer. Therefore, the changes the epigenomic landscape due to incorporation of differential expression H3 variant contributes to change in gene expression pattern and thereby contributing to pathogenesis of cancer.

Differential Expression of EZH2 and H3K27me3 in Oral Verrucous Carcinoma and Oral Verrucous Hyperplasia.

Enhancer of zeste homolog 2 (EZH2), a component of the polycomb repressive complex 2 that catalyzes trimethylation of H3K27 (H3K27me3), has been shown to promote tumor development and progression. Expression of EZH2 is associated with cell cycle regulation and cell proliferation in various neoplasms. Oral verrucous hyperplasia (OVH) and Oral verrucous carcinoma (OVC) are rare entities and share several clinical and histopathologic features. Problems distinguishing these lesions are added by a lack of adjacent normal tissue of the biopsy samples and poorly oriented tissue sections. The aim of this study was to investigate the expression of EZH2 and H3K27me3 in OVH and OVC and comparing the expression with normal oral mucosa and oral squamous cell carcinoma (OSCC). Seventy-eight samples, including 25 cases of OVC, 8 cases of OVH, 35 cases of OSCC and 10 cases of normal oral mucosa, were retrieved and submitted for immunohistochemical staining. The results demonstrated that the mean labeling indices (LIs) of EZH2 and H3K27me3 expression were highest in OSCC, followed by the OVC, OVH, and normal mucosa. Statistical differences in EZH2 LI were observed among these lesions whereas H3K27me3 LI was significantly different among OSCC, OVH and normal mucosa. EZH2 LI was found to have a sensitivity of 72.00% and specificity of 87.50% in distinguishing OVH from OVC, and a sensitivity of 57.14% and specificity of 84.00% in distinguishing OVC from OSCC. A positive correlation between EZH2 and H3K27me3 expression was significantly found in OVC but not in OVH and OSCC. These findings highlight the involvement of epigenetic regulation by EZH2-mediated H3K27me3 in the pathogenesis of OVH and OVC, and EZH2 expression indicates disease progression of these verrucous lesions. Diagnostic test analysis further suggests that EZH2 may be used as an additional test for differentiating OVH from OVC in questionable cases.

Histone Variant H2A.J Marks Persistent DNA Damage and Triggers the Secretory Phenotype in Radiation-Induced Senescence.

Irreparable double-strand breaks (DSBs) in response to ionizing radiation (IR) trigger prolonged DNA damage response (DDR) and induce premature senescence. Profound chromatin reorganization with formation of senescence-associated heterochromatin foci (SAHF) is an essential epigenetic mechanism for controlling the senescence-associated secretory phenotype (SASP). To decipher molecular mechanisms provoking continuous DDR leading to premature senescence, radiation-induced DSBs (53BP1-foci) and dynamics of histone variant H2A.J incorporation were analyzed together with chromatin re-modeling in human fibroblasts after IR exposure. High-resolution imaging by transmission electron microscopy revealed that persisting 53BP1-foci developed into DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), consistently located at the periphery of SAHFs. Quantitative immunogold-analysis by electron microscopy revealed that H2A.J, steadily co-localizing with 53BP1, is increasingly incorporated into DNA-SCARS during senescence progression. Strikingly, shRNA-mediated H2A.J depletion in fibroblasts modified senescence-associated chromatin re-structuring and abolished SASP, thereby shutting down the production of inflammatory mediators. These findings provide mechanistic insights into biological phenomena of SASP and suggest that H2A.J inhibition could ablate SASP, without affecting the senescence-associated growth arrest.

Altered expression of epigenetic modifiers EZH2, H3K27me3, and DNA methyltransferases in meningiomas - prognostic biomarkers for routine practice.

INTRODUCTION: Meningiomas comprises of a wide variety of histological entities with heterogeneous biological behaviour and prognosis. The plethora of genetic data are yet to produce relevant biomarkers for routine use. In contrast, epigenetic alterations are less elucidated. MATERIAL AND METHODS: The expression of the key molecules involved in the two principal epigenetic systems, i.e. DNA methylation (DNA methyltransferases [DNMT-1, -3A and -3B]) and histone modification (Enhancer of Zeste homolog-2 [EZH2] and trimethyl histone-3 [H3K27me3]) were assessed in 149 cases of meningiomas (grade I - 102, grade II - 47) by immunohistochemistry. RESULTS: Immunopositivity for EZH2 (38.3% vs. 6.0%) and negativity for H3K27me3 (10.6% vs. 1.0%) were significantly more common in grade II tumours. Both were associated with significantly higher proliferative activity. The majority of the cases of both grades showed expression of all three DNMTs. However, high expression of DNMT-1 was significantly more common in grade II tumours (87.8% vs. 66.2%). Expression of EZH2 and loss of H3K27me3 were associated with significantly shorter progression-free survival (hazard ratio [HR] = 4.07 and 0.24, respectively). CONCLUSIONS: The key epigenetic regulators play important roles in the pathobiology of meningiomas. EZH2 positivity and H3K27me3 negativity are associated with aggressive tumour-biology and poor prognosis. Both these markers can easily be assessed by immunohistochemistry and can be incorporated in routine practice.

Assessment of tumour proliferation by use of the mitotic activity index, and Ki67 and phosphohistone H3 expression, in early-stage luminal breast cancer.

AIMS: Phosphohistone H3 (PhH3) has been proposed as a novel proliferation marker in breast cancer. This study compares the interobserver agreement for assessment of the mitotic activity index (MAI), Ki67 expression, and PhH3 in a cohort of oestrogen receptor (ER)-positive breast cancer patients. METHODS AND RESULTS: Tumour samples of 159 luminal breast cancer patients were collected. MAI and PhH3 scores were assessed by three breast cancer pathologists. Ki67 scores were assessed separately by two of the three pathologists. PhH3-positive cells were counted in an area of 2 mm2 , with a threshold of ≥13 positive cells being used to discriminate between low-proliferative and high-proliferative tumours. Ki67 expression was assessed with the global scoring method. Ki67 percentages of <20% were considered to be low. The intraclass correlation coefficient (ICC) and Cohen's κ statistics were used to evaluate interobserver agreement. The impact on histological grading of replacing the MAI with PhH3 was assessed. Counting PhH3-positive cells was highly reproducible among all three observers (ICC of 0.86). The κ scores for the categorical PhH3 count (κ = 0.78, κ = 0.68, and κ = 0.80) reflected substantial agreement among all observers, whereas agreement for the MAI (κ = 0.38, κ = 0.52, and κ = 0.26) and Ki67 (κ = 0.55) was fair to moderate. When PhH3 was used to determine the histological grade, agreement in grading increased (PhH3, κ = 0.52, κ = 0.48, and κ = 0.52; MAI, κ = 0.43, κ = 0.35, and κ = 0.32), and the proportion of grade III tumours increased (14%, 18%, and 27%). CONCLUSION: PhH3 seems to outperform Ki67 and the MAI as a reproducible means to measure tumour proliferation in luminal-type breast cancer. Variation in the assessment of histological grade might be reduced by using PhH3, but would result in an increase in the proportion of high-grade cancers.

Expression of H3K4me3 and H3K9ac in breast cancer.

PURPOSE: Breast cancer is the leading cause of cancer death in females. Histone modifications have been shown to have an influence on the gene expression. This study focusses on the histone modifications H3K9ac and H3K4me3 in breast cancer and their impact on survival METHODS: H3K4me3 and H3K9ac expression was immunohistochemically examined in 235 tissue samples. RESULTS: Positive estrogen receptor status was correlated with a higher IRS of the nuclear (p = 0.033), and of the cytoplasmic H3K4me3 staining (p = 0.009). H3K9ac intensity was associated to the Her2 status (p = 0.045) and to poor prognosis in cells with positive Ki67 status (p = 0.013). A high intensity of nuclear H3K4me3 staining was found to be correlated with a lower 10-year-survival (p = 0.026) and with lower breast cancer-specific survival (p = 0.004). High percentage score (> 190) of H3K9ac expression was correlated to worse breast cancer-specific survival (p = 0.005). Shorter progression-free survival was found in patients with nuclear (p = 0.013) and cytoplasmic H3K4me3expression (p = 0.024) and H3K9ac expression (p = 0.023). CONCLUSION: This analysis provides new evidence of histone modifications in breast cancer. High H3K4me3 and H3K9ac expression was correlated with survival rates. Further investigation of histone modifications in breast cancer could lead to a more profound understanding of the molecular mechanisms of cancer development and could result in new therapeutic strategies.

Low doses of deoxynivalenol inhibit the cell migration mediated by H3K27me3-targeted downregulation of TEM8 expression.

Deoxynivalenol (DON) is a mycotoxin produced by multipleFusariumspecies that often contaminates cereals and threatens human and animal health. A wide range of cytotoxic effects, such as the induction of DNA damage, an increase in mitochondrial permeability and the inhibition of macromolecule synthesis, have been reported. However, the effects of DON on cell migration-a fundamental process in living cells critical for normal development, immune responses, and disease processes-and the mechanism underlying these effects are still unclear. Here, we showed that DONsignificantly inhibited the migration of MRC-5, CCD-18Co, HCT116 and WM793 cells at 50 ng/ml, 50 ng/ml, 400 ng/ml and 250 ng/ml, respectively, which maintained cell viability at 90%. Further analysis showed that DON inhibited the expression of tumour endothelial marker 8 (TEM8), a key gene in cell migration. Furthermore, we showed that DON inhibited the expression of TEM8 through increasing the level of H3K27me3 in the TEM8 promoter. Finally, overexpression of TEM8 or treating by H3K27me3-specific inhibitor GSK126 attenuated the inhibitory effect of DON on cell migration. In summary, low doses of DON at approximately dietary exposure significantly inhibited cell migration by downregulating the expression of TEM8 in a manner mediated by H3K27me3, which may generate increasing concerns for the risk of DON exposure.

Semisynthesis and Reconstitution of Nucleosomes Carrying Asymmetric Histone Modifications.

Nucleosomes, the basic unit of chromatin, contain a protein core of histone proteins, which are heavily posttranslationally modified. These modifications form a combinatorial language which defines the functional state of the underlying genome. As each histone type exists in two copies in a nucleosome, the modification patterns can differ between the individual histones, resulting in asymmetry and increasing combinatorial complexity. To systematically explore the regulation of chromatin regulatory enzymes (writers, erasers, or readers), chemically defined nucleosomes are required. We have developed strategies to chemically modify histones and control nucleosome assembly, thereby enabling the reconstitution of asymmetric histone modification patterns. Here, we report a detailed protocol for the modular assembly of such nucleosomes. Employing a three-segment ligation strategy for the semisynthesis of H3, coupled with the use of the protease cleavable "lnc-tag," we provide an efficient and traceless method for the controlled semisynthesis and reconstitution of asymmetrically modified nucleosomes.

Histone Acetyltransferase-Dependent Pathways Mediate Upregulation of NADPH Oxidase 5 in Human Macrophages under Inflammatory Conditions: A Potential Mechanism of Reactive Oxygen Species Overproduction in Atherosclerosis.

Histone acetylation plays a major role in epigenetic regulation of gene expression. Monocyte-derived macrophages express functional NADPH oxidase 5 (Nox5) that contributes to oxidative stress in atherogenesis. The mechanisms of Nox5 regulation are not entirely elucidated. The aim of this study was to investigate the expression pattern of key histone acetyltransferase subtypes (p300, HAT1) in human atherosclerosis and to determine their role in mediating the upregulation of Nox5 in macrophages under inflammatory conditions. Human nonatherosclerotic and atherosclerotic tissue samples were collected in order to determine the expression of p300 and HAT1 isoforms, H3K27ac, and Nox5. In vitro determinations were done on human macrophages exposed to lipopolysaccharide in the absence or presence of histone acetyltransferase inhibitors. Western blot, immunohistochemistry, immunofluorescence, real-time PCR, transfection, and chromatin immunoprecipitation assay were employed. The protein levels of p300 and HAT1 isoforms, H3K27ac, and Nox5 were found significantly elevated in human atherosclerotic specimens. Immunohistochemistry/immunofluorescence staining revealed that p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins were colocalized in the area of CD45+/CD68+ immune cells and lipid-rich deposits within human atherosclerotic plaques. Lipopolysaccharide induced the levels of HAT1, H3K27ac, H3K9ac, and Nox5 and the recruitment of p300 and HAT1 at the sites of active transcription within Nox5 gene promoter in cultured human macrophages. Pharmacological inhibition of histone acetyltransferase significantly reduced the Nox5 gene and protein expression in lipopolysaccharide-challenged macrophages. The overexpression of p300 or HAT1 enhanced the Nox5 gene promoter activity. The histone acetyltransferase system is altered in human atherosclerosis. Under inflammatory conditions, HAT subtypes control Nox5 overexpression in cultured human macrophages. The data suggest the existence of a new epigenetic mechanism underlying oxidative stress in atherosclerosis.

gamma-H2AX: A potential biomarker in breast cancer.

Histone H2AX undergoes phosphorylation as an answer to DNA double-strand breaks, which in turn are part of the oncogenic procedure. The detection of gamma-H2AX can potentially serve as a biomarker for transformation of normal tissue to premalignant and consequently to malignant tissues. The aim of this study was to evaluate the clinical significance of gamma-H2AX expression in breast cancer. Gamma-H2AX expression in tissues from 110 breast cancer patients was analyzed by immunohistochemistry and correlated with clinicopathological variables. Greater tumor size, higher grade, and the number of affected lymph nodes are significantly associated with greater values of gamma-H2AX. In addition, gamma-H2AX differs significantly among patients' International Federation of Gynecology and Obstetrics stage. Higher values of estrogen receptor and progesterone receptor are significantly associated with lower gamma-H2AX values. In conclusion, a positive association between gamma-H2AX expression and infaust histopathological parameters was observed.

DNA damage induction during localized chronic exposure to an insoluble radioactive microparticle.

Insoluble radioactive microparticles emitted by the incident at the Fukushima nuclear power plant have drawn keen interests from the viewpoint of radiation protection. Cs-bearing particles have been assumed to adhere in the long term to trachea after aspirated into respiratory system, leading to heterogeneous dose distribution within healthy tissue around the particles. However, the biological effects posed by an insoluble radioactive particle remain unclear. Here, we show cumulative DNA damage in normal human lung cells proximal and distal to the particle (ß-ray and γ-ray-dominant areas, respectively) under localized chronic exposure in comparison with uniform exposure. We put a Cs-bearing particle into a microcapillary tip and placed it onto a glass-base dish containing fibroblast or epithelial cells cultured in vitro. A Monte Carlo simulation with PHITS code provides the radial distribution of absorbed dose-rate around the particle, and subsequently we observed a significant change in nuclear γ-H2AX foci after 24 h or 48 h exposure to the particle. The nuclear foci in the cells distal to the particle increased even under low-dose-rate exposure compared with uniform exposure to 137Cs γ-rays, which was suppressed by a treatment with a scavenger of reactive oxygen species. In contrast, such focus formation was less manifested in the exposed cells proximal to the particle compared with uniform exposure. These data suggest that the localized exposure to a Cs-bearing particle leads to not only disadvantage to distal cells but also advantage to proximal cells. This study is the first to provide quantitative evaluation for the spatial distribution of DNA double strand breaks after the heterogeneous chronic exposure to a Cs-bearing particle in comparison with uniform Cs exposure.

MiR-516a-5p inhibits the proliferation of non-small cell lung cancer by targeting HIST3H2A.

The dysregulation of microRNAs (miRNAs) is associated with the pathogenesis of non-small cell lung cancer (NSCLC). However, the mechanisms by which miR-516a-5p contributes to NSCLC remain unclear. The association between miR-516a-5p expression and the clinicopathological characteristics and prognosis in patients with NSCLC was analyzed by The Cancer Genome Atlas (TCGA) data set. The targets of miR-516a-5p were identified by bioinformatic analysis and luciferase report assay. MTT and soft agar assays were conducted to investigate the function of miR-516a-5p in NSCLC cells. We found that the expression of miR-516a-5p was decreased in NSCLC tissues and associated with the age, pathological stage, and tumor size, acting as an independent prognostic factor of tumor recurrence in patients with NSCLC. Restoration of miR-516a-5p inhibited the cell viability and anchorage-independent growth of NSCLC cells, but its inhibitor had the opposite effects. Histone cluster 3 H2A (HIST3H2A) was further identified as a direct target of miR-516a-5p and displayed a negative correlation with miR-516a-5p expression in NSCLC tissues. Overexpression of HIST3H2A reversed the anti-proliferation effects induced by miR-516a-5p and acted as an independent prognostic factor of poor survival in patients with NSCLC. Altogether, our findings demonstrate that miR-516a-5p may function as a tumor suppressive factor in NSCLC cells by targeting HIST3H2A and might represent a potential indicator of tumor recurrence in patients with NSCLC.

Green tea-induced epigenetic reactivation of tissue inhibitor of matrix metalloproteinase-3 suppresses prostate cancer progression through histone-modifying enzymes.

Green tea polyphenols (GTPs) and their major constituent, epigallocatechin-3-gallate (EGCG), have been reported to demonstrate many interesting biological activities, including anticancer properties. Recent studies on prostate cancer provide strong evidence that epigenetic mechanisms are major players in the regulation of matrix metalloproteinases (MMPs) and their binding partner tissue inhibitor of MMPs (TIMPs) involved in prostate cancer progression. Here we demonstrate that GTP/EGCG mediate epigenetic reactivation of TIMP-3 that plays a key role in suppressing invasiveness and cancer progression. Treatment of human prostate cancer DUPRO and LNCaP cells with 10 µg/mL GTP and 20 µM EGCG induced TIMP-3 mRNA and protein expression. This transcriptional activation of TIMP-3 was associated with the decrease in the expression of both enhancers of zeste homolog 2 (EZH2) and its catalytic product trimethylation of histone H3 at lysine 27 (H3K27me3) repressive marks at the TIMP-3 promoter with an accompanying increase in histone H3K9/18 acetylation. In addition, GTP/EGCG treatment significantly reduced class I histone deacetylase (HDAC) activity/expression and EZH2 and H3K27me3 levels in prostate cancer cells. EGCG/GTP exposure also reduced MMP-2/MMP-9 gelatinolytic activity and abrogated invasion and migration capabilities in these cells. Silencing of EZH2 and class I HDACs strikingly increased the expression of TIMP-3 independent of DNA methylation. Furthermore, clinical trials performed on patients undergoing prostatectomy consuming 800 mg EGCG (Polyphenon E) up to 6 weeks and grade-matched controls demonstrate an increase in plasma TIMP-3 levels. A marked reduction in class I HDACs activity/expression and EZH2 and H3K27me3 levels were noted in GTP-supplemented prostate tissue. Our findings highlight that TIMP-3 induction, as a key epigenetic event modulated by green tea in restoring the MMP:TIMP balance suppresses prostate cancer progression.

Expression of γ-H2AX and patient prognosis in breast cancer cohort.

H2AX phosphorylation is a novel marker of DNA double-stranded breaks. In the present study, we assessed the γ-H2AX expression, its association with other clinicopathologic characteristics, and the prognosis in a cohort of 97 patients with breast cancer. Ninety-seven specimens of tumor tissue and 77 adjacent normal tissues from patients with breast cancer were examined. All patients underwent modified radical mastectomy or local tumor resection without lymph node dissection. γ-H2AX expression was assessed by standard immunohistochemistry. Patients were followed after surgery for a mean duration of 70.1 ± 18.7 months (range, 6-93 months). The γ-H2AX staining was positive in 27 (27.8%) patients. The positive rates of H2AX were 26.0% and 2.6% in tumor tissue and adjacent normal tissues, respectively. γ-H2AX positive status was negatively associated with TNM staging, with 24 positive cases (32.4%) in TNM staging I-II, while no positive cases in TNM staging III-IV (P = 0.026). Sixteen patients (16.5%) died during the follow-up. No significant association between γ-H2AX expression and patient survival was detected. The unadjusted HR (hazard ratio) for γ-H2AX positive was 0.84 (95% CI: 0.27, 2.60). In TNM staging subgroup analysis, death only occurred in γ-H2AX negative patients. Our study is the first study to demonstrate that expression of γ-H2AX is associated with TNM staging. Due to the small sample and limited follow-up time, we did not observe a significant association between γ-H2AX and patient survival. γ-H2AX expression could be a potential biomarker for cancer diagnosis and prediction, and further studies are in need.

Mequindox induces apoptosis, DNA damage, and carcinogenicity in Wistar rats.

Mequindox (MEQ) is a synthetic antibacterial agent. Recent studies showed that MEQ and its primary metabolites exhibit strong genotoxicity to mammalian cells, and MEQ induced carcinogenicity in mice. These findings suggest that chronic exposure to MEQ could lead to an increased risk of cancer later in life. In the present study, four groups of Wistar rats (55 rats/sex/group) were fed with diets containing MEQ (0, 25, 55, and 110 mg/kg) for 2 years. The results showed that the hematological system, liver, kidneys, and adrenal glands, as well as the developmental and reproductive systems, were the main targets for MEQ. Liver toxicity mediated by MEQ was associated with apoptosis and the nuclear factor κB (NF-κB) signaling pathway. In addition, MEQ increased the incidence of tumors in rats. Phosphorylated histone H2AX (γ-H2AX) is identified as a biomarker of cellular response to DNA double-strand breaks (DSB). Our data demonstrated that γ-H2AX expression was significantly increased in tumors. Thus, high levels of DSB might be responsible for carcinogenesis in rats, and further investigation is absolutely required to clarify the exact molecular mechanisms for carcinogenicity caused by MEQ in vivo.