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MicroRNAs (miRNAs) are small non-coding RNAs which contribute to the regulation of many physiological and pathological processes. Conventionally, miRNAs perform their activity in the cytoplasm where they regulate gene expression by interacting in a sequence-specific manner with mature messenger RNAs. Recent studies point to the presence of mature miRNAs in the nucleus. This review summarizes current findings regarding the molecular activities of nuclear miRNAs. These molecules can regulate gene expression at the transcriptional level by directly binding DNA on the promoter or the enhancer of regulated genes. miRNAs recruit different protein complexes to these regions, resulting in activation or repression of transcription, through a number of molecular mechanisms. Hematopoiesis is presented as a paradigmatic biological process whereby nuclear miRNAs possess a relevant regulatory role. Nuclear miRNAs can influence gene expression by affecting nuclear mRNA processing and by regulating pri-miRNA maturation, thus impacting the biogenesis of miRNAs themselves. Overall, nuclear miRNAs are biologically active molecules that can be critical for the fine tuning of gene expression and deserve further studies in a number of physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

The present study examined the in vitro effects on oral squamous cell carcinoma cells (HSC-3) of cannabidiol (CBD), the main chemical component of Cannabis, proposed as a novel adjuvant therapy in the treatment of cancers. Cell viability (MTT assay), morphology (SEM), apoptosis and cell cycle (flow cytometry), and DNA damage (phospho-γ-H2AX immunofluorescence) were evaluated. Cytotoxicity was evaluated with concentrations between 100 µM and 1 µM, and two concentrations were selected for subsequent analysis: 25 µM, as toxic dose, and 6.25 µM, as non-toxic. CBD caused a dose- and time-dependent reduction in viability of 64 %, 96 %, and 99 % with 25 µM, 50 µM and 100 µM, respectively, after 72 h (p < 0.001), cell cycle arrest in G0-G1 phase with increased apoptosis in particular at 72 h for 25 µM (p < 0.001), significant morphological alterations with 25 µM, still present even at 6.25 µM, and significantly increased cell damage considering a significant increase in the percentage of highly positive cells (5 phosphorylated γH2AX foci), which is around 29 % for 25 µM and 19 % for 6.25 µM after 24 h. CBD inhibits oral cancer growth causing DNA damage. In general, induced cell cytotoxicity appears to be dose- and time-related. Doses of CBD ≥25 μM showed a high reduction in viability. CBD could possibly represent a new therapeutic molecule for its cytotoxic effects against oral squamous cell carcinoma. The mechanism involved in the suppressive effect caused by CBD needs further investigation. [Display omitted] • CBD induced dose- and time-related cytotoxicity. • Concentrations of CBD ≥25 μM showed a high reduction in cell viability. • CBD caused DNA damage inhibiting oral cancer cells. • The mechanism involved in CBD suppressive effect needs further investigation. • CBD represent a new therapeutic molecule against oral squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

This systematic review examine the biological effects of CBD, a major component of therapeutic Cannabis, on human pathological and cancer cell populations of integumentary, gastro-intestinal, genital and breast, respiratory, nervous, haematopoietic and skeletal districts in terms of cell viability, proliferation, migration, apoptosis, inflammation, metastasis, and CBD receptor expression. The included studies were in English, on human cell lines and primary culture from non-healthy donors with CBD exposure as variable and no CBD exposure as control. Quality assessment was based on ToxRtool with a reliability score ranging from 15 to 18. Following the PRISMA statement 4 independent reviewers performed an electronic search using MEDLINE via PubMed, Scopus and Web of Science. From 3974 articles, 83 studies have been selected. Data showed conflicting results due to different concentration exposure, administrations and time points. CBD inhibited cell viability and proliferation in most cellular districts except the integumentary apparatus. Also a significant inhibition of migration was observed in all cell types, while an increase in apoptosis at both high and low doses (greater and less than 10 μM respectively). Considering inflammation, CBD caused an anti-inflammatory effect on nervous cells at low doses and on gastro-intestinal cells at high doses, while metastatic power was reduced even at low doses, but in a skeletal cell line there was an increased angiogenesis. CB1 receptor has been related to viability effects, CB2 to apoptosis and TRPV1 to inflammation and invasiveness. A detailed insight into these aspects would allow therapeutic use of this substance without possible side effects. [Display omitted] [ABSTRACT FROM AUTHOR]

Epigenetic alterations, including aberrant DNA methylation, contribute to tumor development and progression. Silencing of tumor suppressor genes may be ascribed to promoter DNA hypermethylation, a reversible phenomenon intensely investigated as potential therapeutic target. Previously, we demonstrated that eicosapentaenoic acid (EPA) exhibits a DNA demethylating action that promotes the re-expression of the tumor suppressor gene CCAAT/enhancer-binding protein δ (C/EBPδ). The C/EBPβ/C/EBPδ heterodimer formed appears essential for the monocyte differentiation commitment. The present study aims to evaluate the effect of EPA on RAS/extracellular signal regulated kinases (ERK1/2)/C/EBPβ pathway, known to be induced during the monocyte differentiation program. We found that EPA conditioning of U937 leukemia cells activated RAS/ERK/C/EBPβ pathway, increasing the C/EBPβ and ERK1/2 active phosphorylated forms. Transcriptional induction of the upstream activator H-Ras gene resulted in increased expression of H-Ras protein in the active pool of non raft membrane fraction. H-Ras gene analysis identified an hypermethylated CpG island in intron 1 that can affect the DNA-protein interaction modifying RNA polymerase II (RNAPII) activity. EPA treatment demethylated almost completely this CpG island, which was associated with an enrichment of active RNAPII. The increased binding of the H-Ras transcriptional regulator p53 to its consensus sequence within the intronic CpG island further confirmed the effect of EPA as demethylating agent. Our results provide the first evidence that an endogenous polyunsaturated fatty acid (PUFA) promotes a DNA demethylation process responsible for the activation of RAS/ERK/C/EBPβ pathway during the monocyte differentiation commitment. The new role of EPA as demethylating agent paves the way for studying PUFA action when aberrant DNA methylation is involved. [ABSTRACT FROM AUTHOR]

Abstract: Estrogen receptors (ERs) are a recognized prognostic factor and therapeutic target in breast cancer. The loss of ER expression relates to poor prognosis, poor clinical outcome and impairs the use of anti-estrogenic treatment. Histone deacetylase inhibitors are candidate drugs for cancer therapy. Among them, valproic acid (VPA) is a long used and safe anti-epileptic drug. We studied the biological consequences of the chromatin remodeling action of VPA in a normal human mammary epithelial cell line and in ERα-positive and ERα-negative breast cancer cell lines. In these cells and regardless of their ER status, VPA-induced cell differentiation, as shown by increased milk lipids production, decreased expression of the CD44 antigen and growth arrest in the G0–G1 phase of the cell cycle. These effects were accompanied by decreased Rb phosphorylation, hyperacetylation of the p21WAF1/CIP1 gene promoter and increased p21 protein expression. Only in breast cancer cells, cyclin B1 expression was decreased and the cells accumulated also in G2. ERα expression decreased in ERα-positive, increased in ERα-negative and was unchanged in normal mammary epithelial cells, as did the expression of progesterone receptor, a physiological ERα target. VPA decreased the expression of the invasiveness marker pS2 in ERα-positive breast cancer cells, but did not cause its re-expression in ERα-negative cells. Overall, these data suggest that in both ERα-positive and -negative malignant mammary epithelial cells VPA reprograms the cells to a more differentiated and “physiologic” phenotype that may improve the sensitivity to endocrine therapy and/or chemotherapy in breast cancer patients. [Copyright &y& Elsevier]

Cannabidiol is gaining increasing interest for its potential anti-inflammatory, immunomodulatory, and antineoplastic effects. The purpose of this study is to investigate the biological effects of acute and chronic CBD administration on gingival fibroblasts and oral keratinocytes. Viability, morphology, migration, apoptosis and cell cycle, and expression of related genes (p53, BCL2 , p21, and BAX) and of endocannabinoid system receptors (CB1, CB2 and GPR55) with real-time PCR and DNA damage with phospho-γ-H2AX immunofluorescence detection were analyzed. Concentrations between 100 μM and 0.001 μM were used: 50 μM (toxic dose), 25 μM (viability promoter), and 1 μM (nontoxic), were selected for subsequent chronic analysis. Acute treatment reveals significant effects than chronic, in particular in fibroblasts: concentrations ≥50 μM are highly cytotoxic, with increased apoptosis and reduced migration. Cell death correlates with increased p53 and BAX , followed by arrest in G0/G1 phase, with elevated p21 levels, suggesting a time- and dose-dependent damage. An increase in H2AX phosphorylation was observed with 25 μM and 50 μM, while 1 μM was biocompatible. Keratinocytes showed less cytotoxic effect than fibroblasts. Induced cell damage was dose- and time-related, with less damage after chronic treatment. Further investigations are needed with longer time frames to evaluate CBD dose- and time-dependent effects to identify an effective therapeutic dose. [Display omitted] • Fibroblasts were more influenced by CBD administrations. • Acute administration show significant effects compared to chronic treatment. • CBD effects are dose- and time-dependent. • ≥ 50 μM highlight high cytotoxicity and apoptosis, and reduced migration. • 1 μM is biocompatible, 25 μM proliferative effect needs further investigation. [ABSTRACT FROM AUTHOR]

In cancer cells, global genomic hypomethylation is found together with localized hypermethylation of CpG islands within the promoters and regulatory regions of silenced tumor suppressor genes. Demethylating agents may reverse hypermethylation, thus promoting gene re-expression. Unfortunately, demethylating strategies are not efficient in solid tumor cells. DNA demethylation is mediated by ten-eleven translocation (TET) enzymes. They sequentially convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is associated with active transcription; 5-formylcytosine; and finally, 5-carboxylcytosine. Although α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid, the major n-3 polyunsaturated fatty acids, have anti-cancer effects, their action, as DNA-demethylating agents, has never been investigated in solid tumor cells. Here, we report that EPA demethylates DNA in hepatocarcinoma cells. EPA rapidly increases 5hmC on DNA, inducing p21Waf1/Cip1 gene expression, which slows cancer cell-cycle progression. We show that the underlying molecular mechanism involves TET1. EPA simultaneously binds peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), thus promoting their heterodimer and inducing a PPARγ-TET1 interaction. They generate a TET1-PPARγ-RXRα protein complex, which binds to a hypermethylated CpG island on the p21 gene, where TET1 converts 5mC to 5hmC. In an apparent shuttling motion, PPARγ and RXRα leave the DNA, whereas TET1 associates stably. Overall, EPA directly regulates DNA methylation levels, permitting TET1 to exert its anti-tumoral function. [ABSTRACT FROM AUTHOR]

The article focuses on investigating the biological effects of acute and chronic CBD administration on gingival fibroblasts and oral keratinocytes, assessing cell viability, morphology, migration, apoptosis, cell cycle, gene expression, endocannabinoid system receptors, and DNA damage.

To investigate the biological effects of electronic cigarette (e-cigarette) and heated tobacco product extracts respect to tobacco smoke extract on human gingival fibroblasts and human oral keratinocytes analysing cell viability, morphology, migration, apoptosis, cell cycle and epithelial-mesenchymal transition (EMT). Human gingival fibroblasts and human oral keratinocytes viability was analysed by MTT assay, cell morphology using scanning electron microscope (SEM) and cell migration by Scratch assay, a method that mimics the cell migration during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry and the related-gene expression of TP53, BCL2, CDKN2A and CDKN1A was indagated using real-time polymerase chain reaction. EMT process was analysed through expression of specific markers: CDH1, SNAI2, TWIST1, MMP2, FN1 and VIM. All investigations were evaluated after 24 h an in vitro exposure. Undiluted tobacco smoke extract induced significant inhibition of cell viability and cell migration, caused morphological alterations and induced an increase in cell death. No alterations or damage were observed after treatment with e-cigarette extracts. Heated tobacco product extract induced proliferation as highlighted by an increase of cell viability, cell migration and alterations of cycle analysis. Comparing the different cigarette extracts, tobacco smoke turns out to be the most harmful, e-cigarette did not determine morphological and functional alterations and heated tobacco product must be carefully investigated for its possible clinical effects on oral cell populations. • The study compares biological profiles of tobacco smoke, e-cigarette, heated tobacco. • Tobacco smoke induced significant damage. • E-cigarette wasn't associated with significant morphological and functional changes. • E-cigarette emerged as less harmful smoking device to oral cell populations. • Heated tobacco product induced a complex pattern of functional changes. [ABSTRACT FROM AUTHOR]

The article presents a study on in vitro effects of electronic cigarette and heated tobacco product (IQOS) on oral cells. Topics include information on smoking related deaths annually in world; biological effects of electronic cigarette (e-cigarette) and heated tobacco product (IQOS) extracts on human gingival fibroblasts and human oral keratinocytes cells; and how cell viability is analysed by MTT assay.

Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoi-esis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin "bivalent domains," hypermethylation, recruitment of polycomb (PcG)-RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes het-erochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs or miRs) regulate diverse normal and abnormal cell functions. We have identified a regulatory pathway in normal megakaryopoiesis, involving the PLZF transcription factor, miR-146a and the SDF-1 receptor CXCR4. In leukaemic cell lines PLZF overexpression downmodulated miR-146a and upregulated CXCR4 protein, whereas PLZF knockdown induced the opposite effects. In vitro assays showed that PLZF interacts with and inhibits the miR-146a promoter, and that miR-146a targets CXCR4 mRNA, impeding its translation. In megakaryopoietic cultures of CD34+ progenitors, PLZF was upregulated, whereas miR-146a expression decreased and CXCR4 protein increased. MiR-146a overexpression and PLZF or CXCR4 silencing impaired megakaryocytic (Mk) proliferation, differentiation and maturation, as well as Mk colony formation. Mir-146a knockdown induced the opposite effects. Rescue experiments indicated that the effects of PLZF and miR-146a are mediated by miR-146a and CXCR4, respectively. Our data indicate that megakaryopoiesis is controlled by a cascade pathway, in which PLZF suppresses miR-146a transcription and thereby activates CXCR4 translation. [ABSTRACT FROM AUTHOR]

The AML1/ETO and PML/RARaleukemia fusion proteins induce acute myeloid leukemia by acting as transcriptional repressors. They interact with corepressors, such as N-CoR and SMRT, that recruit a multiprotein complex containing histone deacetylases on crucial myeloid differentiation genes. This leads to gene repression contributing to generate a differentiation block. We expressed in leukemia cells containing PML/RARaand AML1/ETO N-CoR protein fragments derived from fusion protein/corepressor interaction surfaces. This blocks N-CoR/SMRT binding by these fusion proteins, and disrupts the repressor protein complex. In consequence, the expression of genes repressed by these fusion proteins increases and differentiation response to vitamin D3 and retinoic acid is restored in previously resistant cells. The alteration of PML/RARa-N-CoR/SMRT connections triggers proteasomal degradation of the fusion protein. The N-CoR fragments are biologically effective also when directly transduced by virtue of a protein transduction domain. Our data indicate that fusion protein activity is permanently required to maintain the leukemia phenotype and show the route to developing a novel therapeutic approach for leukemia, based on its molecular pathogenesis. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) is the primary cause of cancer-related death worldwide, with a low 5-year survival rate even in fully resected early-stage disease. Novel biomarkers to identify patients at higher risk of relapse are needed. We studied the prognostic value of 84 circulating microRNAs (miRNAs) in 182 patients with resected early-stage NSCLC (99 adenocarcinoma (ADC), 83 squamous cell carcinoma (SCC)) from whom peripheral blood samples were collected pre-surgery. miRNA expression was analyzed in relation to disease-free survival (DFS) and overall survival (OS). In univariable analyses, five miRNAs (miR-26a-5p, miR-126-3p, miR-130b-3p, miR-205-5p, and miR-21-5p) were significantly associated with DFS in SCC, and four (miR-130b-3p, miR-26a-5p, miR-126-3p, and miR-205-5p) remained significantly associated with OS. In ADC, miR-222-3p, miR-22-3p, and mir-93-5p were significantly associated with DFS, miR-22-3p remaining significant for OS. Given the high-dimensionality of the dataset, multivariable models were obtained using a regularized Cox regression including all miRNAs and clinical covariates. After adjustment for disease stage, only miR-126-3p showed an independent prognostic role, with higher values associated with longer DFS in SCC patients. With regard to ADC and OS, no miRNA remained significant in multivariable analysis. Further investigation into the role of miR-126 as a prognostic marker in early-stage NSCLC is warranted. [ABSTRACT FROM AUTHOR]

Correction to: The EMBO Journal (2005) 24, 1232-1242. doi:10.1038/sj.emboj.7600593 [ABSTRACT FROM AUTHOR]