ABSTRACT
The transcription repressor REST in the dorsal root ganglion (DRG) is upregulated by peripheral nerve injury and promotes the development of chronic pain. However, the genes targeted by REST in neuropathic pain development remain unclear. The expression levels of 4 opioid receptor (Oprm1, Oprd1, Oprl1, Oprk1) and the cannabinoid CB1 receptor (Cnr1) genes in the DRG regulate nociception. In this study, we determined the role of REST in the control of their expression in the DRG induced by spared nerve injury (SNI) in both male and female mice. Transcriptomic analyses of male mouse DRGs followed by quantitative reverse transcription polymerase chain reaction analyses of both male and female mouse DRGs showed that SNI upregulated expression of Rest and downregulated mRNA levels of all 4 opioid receptor and Cnr1 genes, but Oprm1 was upregulated in female mice. Analysis of publicly available bioinformatic data suggested that REST binds to the promoter regions of Oprm1 and Cnr1. Chromatin immunoprecipitation analyses indicated differing levels of REST at these promoters in male and female mice. Full-length Rest conditional knockout in primary sensory neurons reduced SNI-induced pain hypersensitivity and rescued the SNI-induced reduction in the expression of Oprd1 and Cnr1 in the DRG in both male and female mice. Our results suggest that nerve injury represses the transcription of Oprd1 and Cnr1 via REST in primary sensory neurons and that REST is a potential therapeutic target for neuropathic pain.
ABSTRACT
Leaching of the plastic constituents leading to their chronic exposure to humans is a major concern for our environmental and occupational health. Our previous and other numerous studies have demonstrated that environmental chemicals like di (2-Ethylhexyl)-phthalate (DEHP) could pose a risk towards the epigenetic mechanisms. Yet, the mechanisms underlying its possible epigenotoxicity are poorly understood. We aimed to assess the impact of DEHP exposure to the human breast cancer cells (MCF-7) and resultant changes in DNA methylation regulators ultimately altering the expression of the cell cycle regulator p21 as a model gene. The MCF-7 cells were exposed to environmentally relevant concentrations (50-500 nM) for 24 h. The results showed that DEHP was proliferative towards the MCF-7 cells while it induced global DNA hypermethylation with selective upregulation of DNMT1 and MECP2. In addition, DEHP significantly reduced p53 protein and its enrichment to the DNMT1 promoter binding site, while elevating SP1 and E2F1 transcription factor levels, stimulating their binding to the promoter DNA. Coincidently, increased DNMT1 level was highly associated with loss of p21 expression and increased cyclin D1 levels. Importantly, the p21, but not cyclin D1 promoter CpG-dinucleotides were hypermethylated after exposure to 500 nM DEHP for 24 h. Furthermore, it was observed that DEHP significantly enriched DNMT1 and MECP2 to the p21 promoter to induce DNA methylation-based epigenetic silencing of p21, resulting in increased cell proliferation. Our results suggest DEHP could potentially induce the epigenetic alterations that might increase the risk of breast cancer, given that the underlying mechanisms should be fully elucidated.
Subject(s)
Diethylhexyl Phthalate , Phthalic Acids , DNA Methylation , Diethylhexyl Phthalate/toxicity , Humans , Promoter Regions, GeneticABSTRACT
BACKGROUND: Cancer is an outcome of uncontrolled cell division eventually associated with dysregulated epigenetic mechanisms, including DNA methylation. DNA methyltransferase 1 is ubiquitously expressed in the proliferating cells and is essential for the maintenance of DNA methylation. It causes the abnormal silencing of tumor suppressor genes in human cancer which is necessary for proliferation, cell cycle progression, and survival. DNMT1 is involved in tumorigenesis of several cancers, its upregulation potentially upscale the promoter level inactivation of transcription of a tumor inhibitory gene by introducing repressive methylation marks on the CpG islands. This epigenetic perturbation caused by DNMT is targeted for cancer therapeutics. PURPOSE: To demonstrate the proliferative inhibitory potential of brazilin in human breast cancer cell line (MCF-7) with concurrent mitigation of DNMT1 functional expression and to understand its effect on downstream targets like cell cycle inhibitor p21. STUDY DESIGN/ METHODS: The impact of brazilin on the growth and proliferation of the MCF-7 cells was determined using the XTT assay. The global DNA 5-methyl cytosine methylation pattern was analyzed upon brazilin treatment. The gene and protein expression of DNMTs were determined with quantitative RTPCR and western blots respectively. The potential binding sites of transcription factors in the human DNMT1 promoter were predicted using the MatInspector tool on the Genomatix software. The chromatin immunoprecipitation (ChIP) assay was performed to demonstrate the transcription factors occupancy at the promoter. Methylation of promoter CpG islands was determined by the methylation-specific PCR (MSP) upon brazilin treatment. The molecular docking of the human DNMT1 with brazilin (ligand) was performed using the Schrödinger suite. RESULTS: The heterotetracyclic compound brazilin, present in the wood of Caesalpinia sappan, inhibited the proliferation of the human breast cancer cell line (MCF-7) and reduced the DNMT1 expression with a decrease in global DNA methylation. Brazilin, by activating p38 MAPK and elevating p53 levels within the exposed cells. The elevated level of p53 enriched the occupancy at binding sites within 200 bp upstream to the transcription start site in the DNMT1 promoter, resulting in reduced DNMT1 gene expression. Furthermore, the brazilin restored the p21 levels in the exposed cells as the CpGs in the p21 promoter (-128 bp/+17 bp) were significantly demethylated as observed in the methylation-specific PCR (MSP). CONCLUSION: Highly potential anti-proliferative molecule brazilin can modulate the DNMT1 functional expression and restore the cell cycle inhibitor p21expression. We propose that brazilin can be used in therapeutic interventions to restore the deregulated epigenetic mechanisms in cancer.
Subject(s)
Benzopyrans/pharmacology , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA (Cytosine-5-)-Methyltransferase 1 , Epigenesis, Genetic , Tumor Suppressor Protein p53 , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Molecular Docking Simulation , Phytochemicals , Promoter Regions, Genetic , Tumor Suppressor Protein p53/geneticsABSTRACT
Cadmium (Cd) is considered as a carcinogenic chemical with potential to endanger normal cellular functioning. The present study was aimed to investigate the impact of Cd on the expression of two oncogenic epigenetic regulators, viz., protein arginine methyltransferase 5 (PRMT5) and the polycomb repressive complex 2 (PRC2) member enhancer of Zeste homolog 2 (EZH2). Our results indicate that Cd at 1⯵M concentration increases the viability of HepG2 and MCF7 cells and significantly upregulates the expression of PRMT5 and EZH2, leading to an increased global level of symmetric dimethylarginine (SDMA), H4R3me2s, and H3K27me3. The luciferase reporter assay showed that the promoter activity of PRMT5 and EZH2 is significantly enhanced in both cell lines. Furthermore, Cd exposure induces global DNA hypomethylation due to a decrease in DNA methyltransferases (DNMTs) expression. Methylation-specific and bisulfite sequencing PCR reveal that the proximal promoters of PRMT5 and EZH2, which harbour CpG islands, are almost demethylated when exposed to Cd. The Cd exposure also increases the protein level of transcription factors NFYA and E2F1; consistently, the two transcription factors are found to be enriched at the PRMT5 and EZH2 promoter in chromatin immunoprecipitation experiments. The alterations induced by Cd in the two cancer cell lines were also observed in a non-cancerous cell line (HEK-293). In conclusion, we propose that Cd increases the expression of two oncogenic methyltransferases, possibly with a DNA methylation-dependent mechanism. Further studies focused on the epigenetic alterations induced by Cd would provide mechanistic insights on the carcinogenicity of this metal toxicant at the molecular level.
Subject(s)
Cadmium/pharmacology , DNA Methylation/drug effects , Enhancer of Zeste Homolog 2 Protein/metabolism , Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Transcription Factors , CCAAT-Binding Factor/drug effects , CCAAT-Binding Factor/metabolism , Cadmium/metabolism , Cell Survival/drug effects , CpG Islands , E2F1 Transcription Factor/drug effects , E2F1 Transcription Factor/metabolism , Enhancer of Zeste Homolog 2 Protein/drug effects , Enhancer of Zeste Homolog 2 Protein/genetics , HEK293 Cells , Humans , MCF-7 Cells , Methyltransferases/drug effects , Methyltransferases/genetics , Promoter Regions, Genetic/drug effects , Protein-Arginine N-Methyltransferases/drug effects , Protein-Arginine N-Methyltransferases/genetics , Transcription Factors/metabolismABSTRACT
The epigenetic enzymes catalyze posttranslational modifications (PTMs) of histones, which functionally determine gene expression at the chromatin level. Resveratrol (RVT) a much studied anti-cancer natural molecule is known for restoration of BRCA1, p53, and p21 in cancer cells. We aimed to investigate the role of histone methylation and acetylation on upregulation of these tumor suppressor genes. Our results suggest RVT significantly increase expression of BRCA1, p53, and p21, while decreased expression of protein arginine methyltransferase 5 (PRMT5) and enhancer of Zeste homolog 2 (EZH2) at a 20 µM concentration by 48 hr in both MCF-7 and MDA-MB-231 breast cancer cells. Also, there was an overall loss of H4R3me2s (catalytic product of PRMT5) and H3K27me3 (catalytic product of PRMT5). In contrast, RVT exposure caused a significant decrease in lysine deacetylase (KDAC) activity and expression of KDAC1-3, whereas the expression of lysine acetyltransferase KAT2A/3B was increased compared to the unexposed cells. As an outcome, RVT increased global level of H3K9ac and H3K27ac marks. The chromatin immunoprecipitation showed 20 µM RVT exposure significantly reduced the enrichment of repressive histone marks (H4R3me2s and H3K27me3) while the abundance of activating histone marks (H3K9/27ac) within the proximal promoter region of BRCA1, p53, and p21 was increased. We hypothesize RVT by affecting the expression and function of methylation and acetylation enzymes altered the epigenetic modifications on promoter histones that restored expression of these critically important tumor suppressor genes.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , BRCA1 Protein/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Gene Expression Regulation, Neoplastic , Histones/genetics , Resveratrol/pharmacology , Tumor Suppressor Protein p53/genetics , BRCA1 Protein/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Epigenesis, Genetic , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Histone Deacetylase 2/genetics , Histone Deacetylase 2/metabolism , Histones/metabolism , Humans , MCF-7 Cells , Promoter Regions, Genetic , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Aim: To examine the impact of 4-nonylphenol (4-NP), on the expression of polycomb repressive complexes and cellular proliferation. Materials & methods: Cell proliferation assays, quantitative PCR, Western blotting, luciferase reporter assay, chromatin immunoprecipitation-quantitative PCR were used for the study. Results: The 4-NP at 100 nM concentration significantly increased proliferation of MCF-7 cells. It enhanced the expression of RNF2-BMI1 and EZH2-SUZ12 and concomitantly increased H2AK119ub1 and H3K27me3 repressive marks at p21 proximal promoter resulting in its reduced expression. Selective inhibition of RNF2 or EZH2 reverted the 4-NP action. The phospho-CREB, SP1 and E2F-1 are enriched at proximal promoter of RNF2 and EZH2 and cyclin D1, but not p21. Conclusion: The 4-NP-mediated upregulation of RNF2 and EZH2 resulted in epigenetic silencing of p21.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Epigenesis, Genetic , Histones/genetics , Phenols/pharmacology , Polycomb Repressive Complex 1/genetics , Cell Line, Tumor , Cell Proliferation , Humans , Promoter Regions, Genetic/genetics , Up-RegulationABSTRACT
Curcumin, the active component of the spice turmeric, induce global DNA hypomethylation as it has been shown to inhibit DNA methyltransferases. It promotes cell death in cancer cells by arresting in the G1 phase. It was explained to cause increased expression of cell cycle regulator, p21 (WAF1/Cip1); however, the mechanism remains not clear. The p21 promoter harvests a CpG island (CGI) in the proximal region enriched with CG dinucleotide clusters with Kruppel-like factor 4 (KLF4) transcription factor binding site. We probed the p21 promoter CGI (spanning from -135 to +12, respective to the transcription start site) to detect alterations in cytosine methylation level in response to curcumin exposure in four different human cancer cell lines: A431, A549, MCF7, and HeLa. We observed curcumin (20 µM) treatment significantly increased the expression of p21, and the promoter CGI was demethylated in a dose-dependent manner. The curcumin significantly raised the level KLF4 and enhanced the p21 promoter occupancy by KLF4. From our results we hypothesize that curcumin-mediated demethylation of the p21 proximal promoter and increased KLF4 expression as well as its binding to its proximal promoter could serve as a mechanism that could be hypothesized to cause upregulation of p21 in presence of curcumin and thus its therapeutic implications could further be investigated.
Subject(s)
CpG Islands/drug effects , Curcumin/pharmacology , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Methylation/drug effects , Demethylation/drug effects , Kruppel-Like Transcription Factors/metabolism , Plant Extracts/pharmacology , Promoter Regions, Genetic/drug effects , A549 Cells , Binding Sites , Cell Survival/drug effects , Curcuma/chemistry , DNA (Cytosine-5-)-Methyltransferase 1/antagonists & inhibitors , HeLa Cells , Humans , Kruppel-Like Factor 4 , MCF-7 Cells , Signal Transduction/drug effects , Tumor Suppressor Protein p53/metabolismABSTRACT
The protein arginine methyltransferase 5 (PRMT5) and its catalytic partner methylosome protein MEP50 (WDR77) catalyse the mono- and symmetric di-methylation of selective arginines in various histones and non-histone target proteins. It has emerged as a crucial epigenetic regulator in cell proliferation and differentiation; which also reported to be overexpressed in many forms of cancers in humans. In this study, we aimed to assess the modulations in the expression of this enzyme upon exposure to the well-studied natural compound from the spice turmeric, curcumin. We exposed the lung and breast cancer cell lines (A549 and MCF-7) to curcumin (2 and 20 µM) and observed a highly significant inhibitory effect on the expression of both PRMT5 and MEP50. The level of symmetrical dimethylarginine (SDMA) in multiple proteins, and more specifically, the H4R3me2s mark (which predominates in GC-rich motifs in nucleosomal DNA) was also diminished significantly. We also found that curcumin significantly reduced the level and enrichment of the transcription factors Sp1 and NF-YA which shares their binding sites within the GC-rich region of the PRMT5 proximal promoter. Furthermore, the involvement of both PKC-p38-ERK-cFos and AKT-mTOR signalling was observed in reducing the Sp1 and NF-YA expression by curcumin. Therefore, we propose curcumin decreased the expression of PRMT5 in these cells by affecting at least these two transcription factors. Altogether, we report a new molecular target of curcumin and further elucidation of this proposed mechanism through which curcumin affects the PRMT5-MEP50 methyltransferase expression might be explored for its therapeutic application.
Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , CCAAT-Binding Factor/metabolism , Curcumin/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Protein-Arginine N-Methyltransferases/biosynthesis , Sp1 Transcription Factor/metabolism , A549 Cells , Humans , MCF-7 CellsABSTRACT
The studies on lead (Pb) exposure linking to epigenetic modulations are caused by its differential actions on global DNA methylation and histone modifications. These epigenetic changes may result in increased accessibility of the transcription factors to promoter DNA-binding elements leading to activation and expression of the gene. The protein arginine methyltransferase 5 (PRMT5) and its partner methylosome protein 50 (MEP50) together catalyze the mono- and symmetric dimethylation of arginine residues in many histone and non-histone protein substrates. Moreover, it is overexpressed in many forms of cancer. In the present study, the effects of Pb on the PRMT5 and MEP50 expression and formation of the symmetrically dimethylated arginine (SDMA), the catalytic product of the PRMT5-MEP50 complex were analyzed in vitro after exposing the A549 and MCF-7 cells. The results show that exposure to 0.1 and 1â µM of Pb strongly enhanced the expression of both PRMT5 and MEP50 transcript and protein leading to increased SDMA levels globally with H4R3 being increasingly symmetrically dimethylated in a dose-dependent manner after 48â h of Pb exposure in both cell types. The methylation-specific PCR also revealed that the CpG island present on the PRMT5 promoter proximal region was increasingly demethylated as the dose of Pb increased in a 48-h exposure window in both cells, with MCF-7 being more responsive to Pb-mediated PRMT5 promoter demethylation. The bisulfite sequencing confirmed this effect. The findings therefore indicate that Pb exposure increasing the PRMT5 expression might be one of the contributing epigenetic factors in the lead-mediated disease processes as PRMT5 has a versatile role in cellular functions and oncogenesis.
Subject(s)
CpG Islands , DNA Demethylation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Lead/toxicity , Promoter Regions, Genetic , Protein-Arginine N-Methyltransferases/biosynthesis , A549 Cells , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Humans , MCF-7 Cells , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein-Arginine N-Methyltransferases/genetics , Time FactorsABSTRACT
Environmental cues and chemicals can potentially modulate the phenotypic expression of genome through alterations in the epigenetic mechanisms. Endosulfan is one of the extensively used organochlorine pesticides around the world which is known for its endocrine, neuro- and reproductive toxicity. This study was aimed to investigate the potential of α-endosulfan in modulation of multiple epigenetic enzymes in MCF-7 cells. The cells were treated with DMSO (control) or α-endosulfan (1 and 10µM) and the expression of various epigenetic enzymes was assayed by real-time PCR and immunoblotting, in addition to their activity assays. The results shows α-endosulfan, at 1 and 10µM concentration, significantly promoted viability of MCF-7 cells compared to untreated cells after 24h. The expression of DNA methyltransferases (DNMTs) was upregulated while the global DNA methylation status was initially affected, but later recovered. Total intracellular histone deacetylase (HDAC) activity was found to be significantly increased which was correlated with upregulation of class I HDACs (HDAC 1 and 3) while no significant alteration in the other HDAC classes was observed. The expression and activity of arginine and lysine methylation enzymes, protein arginine methyltransferase 5 (PRMT5) and Enhancer of Zeste homolog 2 (EZH2), respectively, were also found to be modulated by α-endosulfan. We found increased expression of histones H3 and H4, trimethylated H3K27 (product of EZH2), symmetric dimethylation of H4R3 (product of PRMT5) and five different (unidentified) proteins whose arginine residues are symmetrically dimethylated (by increased level of PRMT5) were enhanced in response to 10µM α-endosulfan after 24h exposure window. Moreover, overexpression of basal level of estrogen receptor alpha (ERα), suggests estrogenicity of α-endosulfan. In summary, our results shows modulatory impact of α-endosulfan on multiple cellular epigenetic regulators, known to possess oncogenic potential which might contribute to mechanistic insight of its action in future.
Subject(s)
Carcinogenesis/drug effects , Endosulfan/toxicity , Epigenesis, Genetic , Pesticides/toxicity , Enhancer of Zeste Homolog 2 Protein/metabolism , Estrogen Receptor alpha/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Humans , MCF-7 Cells , Protein-Arginine N-Methyltransferases/metabolismABSTRACT
Lectins are omnipresent in almost all life forms, being the proteins which specifically bind to carbohydrate moieties on the cell surface; they have been explored for their anti-tumour activities. In this study, we purified a fucose specific-lectin (IFL) from Fenneropenaeus indicus haemolymph using fucose-affinity column and characterized for its haemagglutination activity, carbohydrate specificity, dependency on cations and cytotoxicity against cancer cells. The lectin showed non-specificity against human erythrocytes. It was a Ca2+-dependent lectin which remained stable over wide pH and temperature ranges. The lectin showed effective dose dependent cytotoxicity against different human cancer cell lines and induced apoptosis in MCF-7 cells as evidenced by DNA ladder assay and PARP cleavage in a dose dependent manner. Moreover, an increased p21 level corresponding to cyclin D downregulation in response to IFL treatment was observed which might work as probable factors to inhibit cell growth and induce apoptosis of MCF-7 cells. Therefore, we report a novel lectin from the prawn haemolymph with high specificity for L-fucose and antiproliferative towards human cancer cells. However, further establishment of the modus operandi of this lectin is required to enable its biotechnological applications.