RESUMO
Lectins or agglutinins are mainly proteins or glycoproteins, reported to uphold an ability to agglutinate the red blood cells (RBCs) with a known sugar specificity in a diverse group of organisms. In the present study, we purified a hemocyanin (named as MmHc) from a shrimp, Metapenaeus monoceros by size-exclusion chromatography. Further characterization revealed that the purified MmHc showed hemagglutination activity that was found to be specifically inhibited by Lewis B and Lewis Y tetrasaccharides. The MmHc displayed two oligomers of molecular weight approximately â¼78 and â¼85 kDa in SDS-PAGE. The native molecular mass of MmHc was found to be â¼457 kDa as determined by size-exclusion chromatography which indicated that the purified MmHc is an oligomeric protein. MmHc showed a maximum activity within pH 7.0-8.0, while a wide range of temperature stability was observed between 4 to 55 °C, however, it did not show any dependency on metal ions for binding. Subsequently, the analysis of the peptides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) identified the purified MmHc as shrimp hemocyanin showing significant similarity to the hemocyanin of Penaeus vannamei. The results of multiple sequence alignment and detailed analysis of the molecular interactions predicted by AutoDock suggested that besides the oxygen carrier function, this MmHc may have multiple roles and can interact well with the Lewis Y antigen through a typical sugar binding motif containing the similar hydrophilic amino acids as the conserved residues.
Assuntos
Penaeidae , Animais , Penaeidae/metabolismo , Hemocianinas/química , Hemocianinas/metabolismo , Hemolinfa/química , Hemolinfa/metabolismo , Lectinas/farmacologia , Lectinas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Açúcares/análiseRESUMO
There has been growing interest on probiotics to enhance weight gain and disease resistance in young calves and to improve the milk yield in lactating animals by reducing the negative energy balance during the peak lactation period. While it has been well established that probiotics modulate the microbial community composition in the gastrointestinal tract, and a probiotic-mediated homeostasis in the rumen could improve feed conversation competence, volatile fatty acid production and nitrogen flow that enhances the milk composition as well as milk production, detailed changes on the molecular and metabolic level prompted by probiotic feed additives are still not understood. Moreover, as living biotherapeutic agents, probiotics have the potential to directly change the gene expression profile of animals by activating the signalling cascade in the host cells. Various direct and indirect components of probiotic approaches to improve the productivity of dairy animals are discussed in this review.
RESUMO
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.
Assuntos
Dietilexilftalato , Ácidos Ftálicos , Metilação de DNA , Dietilexilftalato/toxicidade , Humanos , Regiões Promotoras GenéticasRESUMO
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.
Assuntos
Benzopiranos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , DNA (Citosina-5-)-Metiltransferase 1 , Epigênese Genética , Proteína Supressora de Tumor p53 , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Compostos Fitoquímicos , Regiões Promotoras Genéticas , Proteína Supressora de Tumor p53/genéticaRESUMO
The potent environmental toxicant aflatoxin B1 (AFB1), is a group I carcinogen reported to induce the expression of many cancer associated proteins. Epigenetic alterations such as DNA methylation and histone modifications play vital role in AFB1-mediated carcinogenesis. These epigenetic modifications may result in the recruitment of specific proteins and transcription factors to the promoter region and regulate gene expression. Here we show that AFB1, at lower concentrations (100 and 1000 nM) induced proliferation in L-132 and HaCaT cells with activation of the Akt pathway, which ultimately steered abnormal proliferation and transmission of survival signals. We demonstrated a significant reduction in the expression of p21 with a remarkable increase in the expression of cyclin D1 that correlated with increased methylation of CpG dinucleotides in p21 proximal promoter, while cyclin D1 promoter remained unmethylated. The chromatin immunoprecipitation results revealed the enrichment of DNMT3a and H3K27me3 repressive marks on the p21 proximal promoter where EZH2 mediated H3K27me3 mark enhanced the binding of DNMT3a at the promoter and further contributed to the transcriptional inactivation. The overall study provided the novel information on the impact of AFB1 on p21 inactivation via EZH2 and promoter methylation which is known to be a vital process in proliferation. Furthermore, AFB1 induced the expression of EZH2 analogue protein E(z), cyclin D1 analogue cyclin D and decreased the expression of p21 analogue Dacapo in Drosophila melanogaster. Interestingly, the aggressiveness in their expression upon re-exposure in successive generations suggested first hand perspectives on multigenerational epigenetic memory.
Assuntos
Aflatoxina B1 , Histonas , Aflatoxina B1/toxicidade , Animais , Metilação de DNA , Drosophila melanogaster , Epigênese Genética , Histonas/metabolismoRESUMO
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.
Assuntos
Cádmio/farmacologia , Metilação de DNA/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores de Transcrição , Fator de Ligação a CCAAT/efeitos dos fármacos , Fator de Ligação a CCAAT/metabolismo , Cádmio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ilhas de CpG , Fator de Transcrição E2F1/efeitos dos fármacos , Fator de Transcrição E2F1/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Células HEK293 , Humanos , Células MCF-7 , Metiltransferases/efeitos dos fármacos , Metiltransferases/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteína BRCA1/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Resveratrol/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética , Histonas/genética , Fenóis/farmacologia , Complexo Repressor Polycomb 1/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Regiões Promotoras Genéticas/genética , Regulação para CimaRESUMO
Aflatoxins are fungal metabolites classified into four major groups such as B1, B2, G1, and G2. These natural aflatoxins are designated as group I carcinogen by the International Agency for Research on Cancer. Among these, the aflatoxin B1 is more potent. Protein arginine methyltransferase 5, an epigenetic modulator, emerged as an oncoprotein, is overexpressed in diverse forms of cancers. The present study aims to explore the AFB1-mediated overexpression of PRMT5. The AFB1 at nanomolar concentrations increased the cell viability, as well as the expression of PRMT5 and its binding partner methylosome protein 50 level significantly in L-132 and HaCaT cells. The knockdown of PRMT5 by its siRNA is attenuated by AFB1, thus substantiating AFB1-mediated PRMT5 overexpression. The PKC isoform-specific inhibitor study revealed direct relation with PKCα and an inverse relation with PKCδ. The analysis of mitogen-activated protein kinase pathway revealed reduced p38 phosphorylation with increased phosphorylation of ERK1/2 upon exposure to AFB1. The combination of MEK and PKC inhibitors with AFB1 treatment revealed that PKCα activates downstream kinase ERK which leads to overexpression of PRMT5. In summary, we propose that PKCα and extracellular signal-regulated kinases are conjointly involved in the induction of PRMT5 upon AFB1 exposure.
Assuntos
Aflatoxinas/toxicidade , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Quinase C/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Ilhas de CpG/efeitos dos fármacos , Curcumina/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Metilação de DNA/efeitos dos fármacos , Desmetilação/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/metabolismo , Extratos Vegetais/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Células A549 , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Curcuma/química , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Células HeLa , Humanos , Fator 4 Semelhante a Kruppel , Células MCF-7 , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Fator de Ligação a CCAAT/metabolismo , Curcumina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/biossíntese , Fator de Transcrição Sp1/metabolismo , Células A549 , Humanos , Células MCF-7RESUMO
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.
Assuntos
Ilhas de CpG , Desmetilação do DNA/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Chumbo/toxicidade , Regiões Promotoras Genéticas , Proteína-Arginina N-Metiltransferases/biossíntese , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Células MCF-7 , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Fatores de TempoRESUMO
The compulsive and insidious secondary metabolite aflatoxin B1, produced by the opportunistic fungi Aspergillus flavus, upholds a distinguished place in midst of the toxicants causing fatal hazards to humans. Aflatoxins alter the function of host cells by inducing multiple effects through genetic and non-genetic pathways. Epigenetic mechanisms drag major attention towards finding novel and new mechanisms involved in this process. Our present work intends to study the functional expression profile of multiple epigenetic regulators. AFB1 modulates multiple epigenetic regulators like DNA methyltransferases (DMNTs), histones modifying enzymes and polycomb proteins. AFB1 upregulates the expression of DNMTs at gene and protein level in a dose dependent manner. It reduced the histone acetyl transferase (HAT) activity significantly with a remarkable increase in histone deacetylase (HDAC) activity along with an induction in expression of HDACs gene and protein in a dose dependent manner. The gene and protein expression of polycomb repressor proteins B cell specific moloney murine leukemia virus integration site 1 (BMI-1) and enhancer of zeste homolog 2 (EZH2) was significantly over expressed with enhanced trimethylation of H3K27 and ubiquitination of H2AK119. In summary, our results show impact of aflatoxin B1 on multiple epigenetic modulations known to be pivotal in oncogenic processes.
Assuntos
Aflatoxina B1/toxicidade , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Queratinócitos/efeitos dos fármacos , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismoRESUMO
Study was conducted to determine the biochemical constituents in coconut (Cocos nucifera L.) haustorium, a spongy tissue formed during coconut germination. Results indicated that 100g of dried coconut haustorium contained 1.05±0.2% ash, 44.2±4.6% soluble sugar, 24.5±3.2% starch, 5.50±0.3% protein, 1.99±0.9% fat, 5.72±0.4% soluble dietary fibre, 20.3±1.9% insoluble dietary fibre, and 146±14.3mg phenolics. Mineral profiling showed that it contained 145±8.6, 104±9.6, 33.9±8.2, 30.9±1.9, 9.45±2.1, 0.292±0.1, 2.53±0.2 and 1.20±0.1mg of K, Mg, Ca, P, Mn, Cu, Fe and Zn, respectively. Antioxidant activity assay indicated that 100g haustorium was equivalent to 1918±173, 170±20.4, 72.8±14.7 and 860±116mg of Trolox as measured by CUPRAC, FRAP, DPPH and ABTS, respectively. Amino acid score indicated that methionine+cysteine (57.6%), phenylalanine+tyrosine (32.6%), leucine (45.7%) and isoleucine (68%) are found less in haustorium. Further studies needed in developing nutritionally balanced formulations using coconut haustorium, which will be useful for lactose intolerant children.
Assuntos
Cocos , Fibras na Dieta , Germinação , Humanos , Fenóis , AmidoRESUMO
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.
Assuntos
Carcinogênese/efeitos dos fármacos , Endossulfano/toxicidade , Epigênese Genética , Praguicidas/toxicidade , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Receptor alfa de Estrogênio/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Células MCF-7 , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
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.
Assuntos
Proteínas de Artrópodes/farmacologia , Neoplasias da Mama/tratamento farmacológico , Citotoxinas/farmacologia , Fucose , Penaeidae/química , Lectinas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Animais , Proteínas de Artrópodes/química , Neoplasias da Mama/metabolismo , Citotoxinas/química , Feminino , Humanos , Células MCF-7 , Lectinas de Plantas/química , Proteínas Inativadoras de Ribossomos/químicaRESUMO
Aspergillus flavus is an ubiquitous, opportunistic fungus responsible to cause invasive fungal allergic diseases, including bronchopulmonary invasive aspergillosis in persons with altered immune function. Lectins have been implicated as interaction mediators between the pathogenic fungi and human host. We isolated L-fucose specific lectin from A. flavus (FFL) and purified it to homogeneity with a combination of ion exchange and hydrophobic interaction chromatography methods. Its hemagglutination activity was significantly inhibited by 125 µM L-fucose as compared to other sugars and sugar derivatives. We, then used human cell line L-132, and U937 cell line to explore the possible cytotoxicity and proinflammatory effect of this fucose-specific lectin. The lectin induced the expression of proinflammatory cytokine interleukin-8 (IL-8) in a dose-dependent manner, and it was found to be associated with the p38 mitogen activated protein kinase (MAPK). The p38MAPK signalling pathway regulates the transcription factor activator protein-1 (AP-1) activity, which is the integration point of many signals that can differentially affect the expression and transcriptional activity of a cell. We observed activation of c-Jun, a critical component of the AP-1 complex, mediated by p38MAPK upon the FFL treatment in L-132 cells. Finally, inhibition of p38MAPK by a specific inhibitor attenuates the c-Jun, suggesting the p38MAPK involvement in the c-Jun activation, which in turn transcriptionally activates the induction of IL-8 in response to the lectin. Thus, this study showed a potential lectin-mediated mechanism to modulate the immune response during host-fungus interactions.
Assuntos
Aspergillus flavus/imunologia , Interleucina-8/biossíntese , Lectinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Aspergillus flavus/química , Linhagem Celular , Expressão Gênica , Humanos , Lectinas/isolamento & purificação , Sistema de Sinalização das MAP QuinasesRESUMO
The exposure of naturally occurring mycotoxins affects human health and play a vital role in cancer initiation and progression. Aflatoxin B1 is a difuranocoumarin mycotoxin, classified as a group I carcinogen. The present study was conducted to assess the effect of aflatoxin B1 on epigenetic regulatory proteins. The protein arginine methyltransferase 5 expression was induced upon aflatoxin B1 treatment in a dose and time dependent manner. Further global arginine methylation was also increased in the same manner. This is the first report showing the induction of epigenetic regulatory protein, protein arginine methyltransferase 5 upon aflatoxin B1 treatment. Further study is required to establish the detailed pathway of PRMT5 induction.
Assuntos
Aflatoxina B1/toxicidade , Proteína-Arginina N-Metiltransferases/genética , Regulação para Cima/efeitos dos fármacos , Linhagem Celular , HumanosRESUMO
PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Derme/metabolismo , Regulação da Expressão Gênica/fisiologia , Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 13 Ativada por Mitógeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Derme/citologia , Humanos , Queratinócitos/citologia , MAP Quinase Quinase 3/genética , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , Proteína Quinase 13 Ativada por Mitógeno/genética , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study, we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132. We show that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G(2), and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a prosurvival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells and confirm that PcG protein levels are regulated by proteasome activity.