RESUMEN
MicroRNAs (miRNAs) are key epigenomic regulators of biological processes in animals and plants. These small non coding RNAs form a complex networks that regulate cellular function and development. MiRNAs prevent translation by either inactivation or inducing degradation of mRNA, a major concern in post-transcriptional gene regulation. Aberrant regulation of gene expression by miRNAs is frequently observed in cancer. Overexpression of various 'oncomiRs' and silencing of tumor suppressor miRNAs are associated with various types of human cancers, although overall downregulation of miRNA expression is reported as a hallmark of cancer. Modulations of the total pool of cellular miRNA by alteration in genetic and epigenetic factors associated with the biogenesis of miRNA machinery. It also depends on the availability of cellular miRNAs from its store in the organelles which affect tumor development and cancer progression. Here, we have dissected the roles and pathways of various miRNAs during normal cellular and molecular functions as well as during breast cancer progression. Recent research works and prevailing views implicate that there are two major types of miRNAs; (i) intracellular miRNAs and (ii) extracellular miRNAs. Concept, that the functions of intracellular miRNAs are driven by cellular organelles in mammalian cells. Extracellular miRNAs function in cell-cell communication in extracellular spaces and distance cells through circulation. A detailed understanding of organelle driven miRNA function and the precise role of extracellular miRNAs, pre- and post-therapeutic implications of miRNAs in this scenario would open several avenues for further understanding of miRNA function and can be better exploited for the treatment of breast cancers.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/terapia , MicroARNs/administración & dosificación , Terapia Molecular Dirigida/métodos , Animales , Neoplasias de la Mama/genética , Manejo de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genéticaRESUMEN
Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system.
Asunto(s)
Núcleo Celular/genética , Genoma/genética , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Animales , Factor de Unión a CCCTC/metabolismo , Embrión de Mamíferos , Ratones , Células Madre Embrionarias de Ratones , Proteínas del Grupo Polycomb/metabolismo , Unión Proteica , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The mammalian genome is complex and presents a dynamic structural organization that reflects function. Organization of the genome inside the mammalian nucleus impacts all nuclear processes including but not limited to transcription, replication and repair, and in many biological contexts such as early development, differentiation and physiological adaptations. However, there is limited understating of how 3D organization of the mammalian genome regulates different nuclear processes. Recent advances in microscopy and a myriad of genomics methods -- ropelled by next-generation sequencing -- have advanced our knowledge of genome organization to a great extent. In this review, we discuss nuclear compartments in general and recent advances in the understanding of how mammalian genome is organized in these compartments with an emphasis on dynamics at the nuclear periphery.
Asunto(s)
Núcleo Celular/ultraestructura , Cromatina/ultraestructura , Genoma/genética , Genómica , Animales , Núcleo Celular/genética , Cromatina/genética , Humanos , Mamíferos/genéticaRESUMEN
Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping.
Asunto(s)
Ensamble y Desensamble de Cromatina , Elementos de Facilitación Genéticos , Proteínas Hedgehog/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Neurogénesis , Neuronas/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Animales , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Ratones , Modelos Genéticos , Neurogénesis/genética , Conformación de Ácido Nucleico , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Aberrant epigenetic modifications are responsible for tumor development and cancer progression; however, readily reversible. Bioactive molecules from diets are promising to cure cancer by modulating epigenetic marks and changing immune response. These compounds specifically target the activity of DNMTs and HDACs to cure various human cancers. In view of this, we investigated the anticancer and epigenetic regulatory activities of an edible-plant Paederia foetida. The efficacy of methanolic extract of P. foetida leaves (MEPL) was tested for the modulation of epigenetic factors in gene silencing, i.e. DNMT and HDAC and expression pattern of certain tumor-suppressor genes. After treatment of prostate cancer cells (PC-3 and DU-145) with MEPL, lupeol and ß-sitosterol; induction of apoptosis, decrease in cellular-viability and inhibition of cellular-migration were noticed. Simultaneously there was inhibition of DNMT1, HDACs and pro-inflammatory, IL-6, IL1-ß, TNF-α and anti-inflammatory, IL-10 genes in cancer and THP1 cell lines. The DNMT1 protein content, enzyme activity and Bcl2 expression decreased significantly; however, expression of E-cadherin (CDH1) and pro-apoptotic gene Bax increased significantly after the treatment of cells with drugs. We conclude plant-derived compounds can be considered to target epigenetic machineries involved with malignant transformation and can open new avenues for cancer therapeutics provoking immune response.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inflamación/metabolismo , Extractos Vegetales/farmacología , Neoplasias de la Próstata , Rubiaceae/química , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Humanos , Inflamación/genética , Masculino , Triterpenos Pentacíclicos , Fitoquímicos , Extractos Vegetales/química , Hojas de la Planta/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , SitoesterolesRESUMEN
Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H2O2) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H2O2 treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H2O2 was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H2O2 induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer.
Asunto(s)
Antígenos CD/genética , Neoplasias de la Mama/genética , Cadherinas/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Factores de Transcripción de la Familia Snail/genética , Antioxidantes/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Butadienos/farmacología , Cadherinas/deficiencia , Línea Celular , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Transición Epitelial-Mesenquimal/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Estimación de Kaplan-Meier , Células MCF-7 , Nitrilos/farmacología , Triterpenos Pentacíclicos/farmacología , Transducción de Señal , Sitoesteroles/farmacología , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
Mixed-lineage leukaemia 1 (MLL1) enzyme plays major role in regulating genes associated with vertebrate development. Cell physiology and homeostasis is regulated by microRNAs in diverse microenvironment. In this investigation we have identified conserved miR-193a target sites within the 3'-UTR of MLL1 gene transcript. Utilizing wild type and mutated 3'-UTR constructs and luciferase reporter assays we have clearly demonstrated that miR-193a directly targets the 3'-UTR region of the MLL1 mRNA. Ectopic expression of miR-193a modulated global H3K4 mono-, di- and tri-methylation levels and affects the expression of CAV1, a gene which is specifically modulated by H3K4me3. To determine the implications of this in vitro finding in aberrant physiological conditions we analyzed prostate cancer tissue samples. In this context miR-193a RNA was undetectable and MLL1 was highly expressed with concomitantly high levels of H3K4me, H3K4me2, and H3K4me3 enrichment in the promoters of MLL1 responsive genes. Finally, we showed that prolonged ectopic expression of miR-193a inhibits growth and cell migration, and induces apoptosis. Thus, while our study unveils amplitude of the epigenome, including miRnome it establishes that; (i) miR-193a directly target MLL1 mRNA, (ii) miR-193a impair MLL1 protein production, (iii) miR-193a reduces the overall methylation marks of the genome.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , MicroARNs/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Neoplasias de la Próstata/genética , Regiones no Traducidas 3' , Caveolina 1/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Cromatina/metabolismo , Regulación hacia Abajo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Metilación , Neoplasias de la Próstata/metabolismoRESUMEN
Microtubule associated tumor suppressor 1 (MTUS1) has been recognized as a tumor suppressor gene in multiple cancers. However, the molecular mechanisms underlying the regulation of MTUS1 are yet to be investigated. This study aimed to clarify the significance of DNA methylation in silencing MTUS1 expression. We report that MTUS1 acts as tumor suppressor in non-small cell lung carcinoma (NSCLC). Analysis of in silico database and subsequent knockdown of DNMT1 suggested an inverse correlation between DNMT1 and MTUS1 function. Interestingly, increased methylation at MTUS1 promoter is associated with low expression of MTUS1. Treatment with DNA methyltransferases (DNMTs) inhibitor, 5-aza-2'-deoxycytidine (AZA) leads to both reduced promoter methylation accompanied with enrichment of H3K9Ac and enhanced MTUS1 expression. Remarkably, knockdown of MTUS1 showed increased proliferation and migration of NSCLC cells in contrast to diminished proliferation and migration, upon treatment with AZA. We concluded that low expression of MTUS1 correlates to DNA methylation and histone deacetylation in human NSCLC.
Asunto(s)
Metilación de ADN/genética , Neoplasias Pulmonares/genética , Proteínas Supresoras de Tumor/genética , Células A549 , Carcinoma de Pulmón de Células no Pequeñas , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor/fisiología , Humanos , Microtúbulos/fisiología , Regiones Promotoras Genéticas/genéticaRESUMEN
Functional analyses of noncoding RNAs have associated many micro RNAs (miRNA, miR) with various physiological processes, including proliferation, differentiation, development, cell metabolism, and apoptosis. Aberrant expression of miRNA and imbalance in their functions may lead to cellular aberration and different disease development, including cancer. In silico analysis of miRNA target prediction suggested that miR-148a possess a binding site in the 3' UTR of DNMT1 mRNA which can cause silencing of DNMT1 gene. Accordingly, we performed in vitro cell culture experiments to confirm the effect miR-148a on DNMT1 gene expression in prostate cancer cell lines. We demonstrated that there is a physical association between DNMT1 mRNA and miR-148a. We found that (i) ectopic expression of miR-148a induces programmed cell death and represses cell proliferation by targeting DNMT1; (ii) miR-148a gene is regulated by DNA methylation and DNMT1 in prostate cancer. We conclude that miR-148a is silenced by DNA methylation and ectopic expression of miR-148a suppresses DNMT1 expression and induced apoptotic genes expression in hormone-refractory prostate cancer cells.
Asunto(s)
Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , MicroARNs/metabolismo , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Próstata/metabolismo , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Supervivencia Celular , ADN (Citosina-5-)-Metiltransferasa 1/genética , Humanos , Masculino , MicroARNs/genética , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/genética , ARN Neoplásico/genéticaRESUMEN
Developmentally inclined hedgehog (HH) signaling pathway and pluripotency inducing transcription factor SOX2 have been known to work syngerstically during cellular reprogramming events to facilitate efficient differentiation. Hence, it is not surprising that both the factors are actively involved in arbitrating malignant growth, including prostate cancer progression. Here, we have described in details the potential mechanisms by which SOX2 effects neoplastic characteristics in prostate cancer and investigated the consequences of simultaneous down-regulation of SOX2 and HH pathway in androgen-independent human prostate cancer cells. Expression of SOX2 has been determined by qRT-PCR, western blot, immunohistochemistry and immunocytochemistry analyses; its functional role determined by gene knockdown using RNAi and over-expression via chemical activation in HaCaT, DU145 and PC-3 cells. Changes in level of cell proliferation, migration and apoptosis profiles were measured by MTT, FACS, chromatin condensation and scratch assays respectively. SOX2 was expressed in all the three cell lines and its inhibition reduced cell proliferation and induced apoptosis. Most importantly, when both SOX2 and HH pathway were targeted simultaneously, cell proliferation was greatly reduced, apoptotic cell population increased drastically and migration potential was reduced. Moreover, gene expression of EMT markers such as E-cadherin and apoptosis related Bcl-2 and Bax was also investigated wherein decrease in E-cadherin and Bcl-2 levels and increase in Bax expression further substantiating our claim. These findings could provide the basis for a novel therapeutic strategy targeting both the effector i.e. SOX2 and perpetuator i.e. HH pathway of aggressive tumorigenic properties in androgen independent prostate cancer.
Asunto(s)
Proteínas Hedgehog/metabolismo , Neoplasias de la Próstata/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Humanos , Masculino , Neoplasias de la Próstata/patologíaRESUMEN
Phospholipase C (PLC)1 is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of ß-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC activation and liposome fusion on the lipid composition.
Asunto(s)
Liposomas/química , Liposomas/metabolismo , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/metabolismo , Dicroismo Circular , Hidrólisis , Metabolismo de los Lípidos , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Soluciones , Espectrometría de Fluorescencia , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
MicroRNAs (miRNA) are small non-coding RNAs which targets most protein-coding transcripts (mRNA) and destroy them. Thus miRNA controls the abundance of those specific proteins and impact on developmental, physiological and pathological processes. Dysregulation of miRNA function thus may lead to various clinicopathological complications, including breast cancer. Silencing of miR-152 gene due to promoter DNA methylation alter the expression pattern of several other genes. E-cadherin (CDH1) forms the core of adherent junctions between surrounding epithelial cells, link with actin cytoskeleton and affects cell signaling. CDH1 gene is down regulated by promoter DNA methylation during cancer progression. In this investigation, we attempt to elucidate the correlation of miR-152 and CDH1 function, as it is well known that the loss of CDH1 function is one of the major reasons for cancer metastasis and aggressiveness of spreading. For the first time we have shown that loss of CDH1 expression is directly proportional to the loss of miR-152 function in breast cancer cells. mRNA and protein expression profile of DNMT1 implicate that miR-152 targets DNMT1 mRNA and inhibits its protein expression. Tracing the molecular marks on DNA and histone 3 for understanding the mechanism of gene regulation by ChIP analyses leads to a paradoxical result that shows DNA methylation adjacent to active histone marking (enrichment of H3K4me3) silence miR-152 gene. Further experiments revealed that DNMT1 plays crucial role for regulation of miR-152 gene. When DNMT1 protein function is blocked miR-152 expression prevails and destroys the mRNA of DNMT1; this molecular regulatory mechanism is creating a cyclic feedback loop, which is now focused as DNMT1/miR-152 switch for on/off of DNMT1 target genes. We discovered modulation of CDH1 gene expression by DNMT1/miR-152 switches. We have demonstrated further that DNMT1 down regulation mediated upregulation of CDH1 (hereafter, DNMT1/CDH1 loop) in presence of ectopic-excess of miR-152 prevents migration of cancer cells. Our data provides novel insights into the regulation mechanism of miRNA and mRNA/protein coding genes and enhances the amplitude of cancer epigenome.
Asunto(s)
Neoplasias de la Mama/genética , Cadherinas/metabolismo , Movimiento Celular/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Histonas/metabolismo , Lisina/metabolismo , MicroARNs/genética , Antígenos CD , Secuencia de Bases , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatina/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , Progresión de la Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/metabolismo , Transfección , Cicatrización de Heridas/genéticaRESUMEN
The role and clinical implication of ZRF1 in breast cancer are poorly understood. So this study is aimed to explore the role of ZRF1 in breast cancer progression. With this context, we first assessed its expression pattern in FFPE primary and metastasis breast tissue samples as well as from publicly available databases. Moreover, we also explored the survival status of patients from the publicly available database and interestingly discover that high expression of ZRF1 decreases the survival of estrogen-positive breast cancer patients more than estrogen-negative status patients. In the perspective of this, we evaluated the role ZRF1 in MCF-7 breast cancer cells and found that it's silencing by knockdown results in decreased cell proliferation as well as cell viability. Results also show that expression of ZRF1 is down regulated in the presence of estrogen-depleted conditions but independent of RAS/MEK as well as AKT axes. Moreover, the decrease in viability of MCF-7 cells was accompanied by induction of apoptosis and DNA damage, well-marked with upregulation of cleaved PARP and downregulation of BCL2 and H2AUbK119 levels. Furthermore, we also explored that knockdown of ZRF1 sensitises the effect of curcumin, observed with decrease in cell viability and dropping of IC50 value from 25 to 15 µM. This investigation thus shed a new light on the role on ZRF1 in breast cancer cells and hence can be exploited to design better therapeutic intervention.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Curcumina/farmacología , Proteínas de Unión al ADN/fisiología , Proteínas Oncogénicas/fisiología , Receptores de Estrógenos/análisis , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/química , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/genética , Femenino , Humanos , Histona Demetilasas con Dominio de Jumonji/análisis , Células MCF-7 , Chaperonas Moleculares , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas de Unión al ARNRESUMEN
BACKGROUND: Caveolin-1 (CAV1) is an important structural component of cellular caveolae involved in cell signaling. CAV1 gene on/off regulatory mechanism in multiple diseases, including cancer is not clearly understood. The tumor suppressor versus oncogene paradox of CAV1 during tumor development tempted us to investigate the role for the epigenetic drift of CAV1 gene regulation. METHODS: We have analyzed CAV1 gene expression and associated epigenetic marks (DNA methylation and histone 3 modifications) in the CAV1 promoter in two colon cancer cell lines, under treatment with well established epigenetic modulators, AZA, SAM, TSA and SFN at varying concentrations. CAV1 gene promoter DNA methylation and histone modifications were analyzed by DNA methylation specific PCR, bisulphite modification of DNA and ChIP analyses following PCR respectively. RESULTS: Ectopic expression of CAV1 by epigenetic modulators inhibits colon cancer cell growth. CAV1 promoter DNA remains unmethylated before and after treatment with epigenetic modulators, which confirmed that DNA methylation is not the regulator of CAV1 expression in colon cancer. There was enrichment of H3K4me3 and H3K9AcS10P and depletion of H3K9me3 modifications around the CAV1 promoter. CONCLUSIONS: Our data provides novel insight into the regulation of CAV1 gene by histone H3 modifications and enhance the amplitude of the cancer epigenome.
Asunto(s)
Caveolina 1/genética , Neoplasias del Colon/genética , Epigénesis Genética , Histonas/metabolismo , Apoptosis , Línea Celular Tumoral , Neoplasias del Colon/patología , Islas de CpG , Epigénesis Genética/efectos de los fármacos , Humanos , Regiones Promotoras GenéticasRESUMEN
Many HDAC inhibitors have passed through the gateway of clinical trials. However, they have limited therapeutic implications due to their pleiotropic pharmaceutical properties and off-target effects. In view of this, dietary active phytochemicals were evaluated. Based upon the chemical and structural insights of HDAC active pockets, thymoquinone (TQ) was investigated to uncover its active participation in HDAC inhibition. The synergistic analysis of docking and molecular dynamics simulation disclosed the elementary interaction and stability of TQ with human HDACs. The in silico findings were corroborated with an in vitro analysis, demonstrating the efficient role of TQ in the attenuation of global HDAC activity. Furthermore, TQ also elicited downstream effects of HDAC inhibition: reactivation of HDAC target genes (p21 and Maspin), induction of the pro-apoptotic gene Bax, down regulation of the anti-apoptotic gene Bcl-2 and arrest of the cell cycle at the G2/M phase. Finally, the result of a higher cytotoxicity of TQ towards MCF-7 breast cancer cells in comparison to normal cells indicates the potential of TQ to be an anticancer drug.
Asunto(s)
Benzoquinonas/química , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Benzoquinonas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Clusterin (CLU) is an important glycoprotein involved in various cellular functions. Different reports have mentioned that the two isoforms of CLU; secretary (sCLU) and nuclear (nCLU) have opposite (paradoxical) roles in cancer development. sCLU provides pro-survival signal, whereas nCLU is involved in pro-apoptotic signaling. However, the molecular mechanism of CLU gene regulation is not clear as of yet. We hypothesize that CLU gene is regulated by DNA methylation and histone modifications and clusterin plays an important role in colon cancer. To evaluate the hypothesis, we investigated CLU expression in colon cancer tissues and DNA methylation and histone modification status of CLU gene promoter. It is apparent from immonohistology data that both benign and cancerous (primary and metastasis) formalin fixed paraffin embedded (FFPE) tissue samples exhibit CLU expression. However and interestingly only noncancerous tissue samples show nCLU expression. Ectopic expression of nCLU either by epigenetic modulators or by nCLU transfection is responsible for colon cancer cell death. To clarify the molecular mechanisms for regulation of expression of CLU isoforms, we have analyzed DNA methylation and histone modifications, such as histone H3K9me3, H3K27me3, H3K4me3, and H3K9AcS10P patterns around the CLU promoter. There is no remarkable change in the DNA methylation status upon treatment of the cells by AZA, TSA and SAM. Our findings clearly show that promoter histone H3K9me3 and H3K27me3 marks are elevated in comparison to H3K4me3 and H3K9AcS10P marks in colon cancer cell lines.
Asunto(s)
Clusterina/genética , Neoplasias del Colon/genética , Histonas/metabolismo , Adulto , Anciano , Secuencia de Bases , Muerte Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clusterina/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Células Tumorales CultivadasRESUMEN
DNA methyltransferases (DNMTs) is a key epigenetic enzyme for pharmacological manipulation and is employed in cancer reprogramming. During past few years multiple strategies have been implemented to excavate epigenetic compounds targeting DNMTs. In light of the emerging concept of chemoinformatics, molecular docking and simulation studies have been employed to accelerate the development of DNMT inhibitors. Among the DNMT inhibitors known till date, epigallocathechin-3-gallate (EGCG) was identified to be effective in reducing DNMT activity. However, the broad spectrum of EGCG to other diseases and variable target enzymes offers some limitations. In view of this, 32 EGCG analogues were screened at S-Adnosyl-L-homocysteine (SAH) binding pocket of DNMTs and procyanidin B2-3, 3'-di-O-gallate (procyanidin B2) was obtained as potent inhibitor having medicinally relevant chemical space. Further, in vitro analysis demonstrates the efficiency of procyanidin B2 in attenuating DNMT activity at IC50 of 6.88±0.647 µM and subsequently enhancing the expression of DNMT target genes, E-cadherin, Maspin and BRCA1. Moreover, the toxic property of procyanidin B2 towards triple negative breast cancer cells to normal cells offers platform for pre-clinical trial and an insight to the treatment of cancer.
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Biflavonoides/farmacología , Catequina/farmacología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proantocianidinas/farmacología , Secuencia de Aminoácidos , Animales , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Catequina/análogos & derivados , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/metabolismo , Femenino , Humanos , Ratones , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
BACKGROUND: DNA methylation mediates gene silencing primarily by inducing repressive chromatin architecture via a common theme of interaction involving methyl-CpG binding (MBD) proteins, histone modifying enzymes and chromatin remodelling complexes. Hence, targeted inhibition of MBD protein function is now considered a potential therapeutic alternative for thwarting DNA hypermethylation prompted neoplastic progress. We have analyzed the gene and protein expression level of the principal factors responsible for gene silencing, that is, DNMT and MBD proteins in MCF-7 and MDA-MB-231 breast cancer cell lines after treatment with various epigenetic drugs. RESULTS: Our study reveals that the epigenetic modulators affect the expression levels at both transcript and protein levels as well as encourage growth arrest and apoptosis in MCF-7 and MDA-MB-231 cells. AZA, TSA, SFN, and SAM inhibit cell growth in MCF-7 and MDA-MB-231 cell lines in a dose-dependent manner, that is, with increasing concentrations of drugs the cell viability gradually decreases. All the epigenetic modulators promote apoptotic cell death, as is evident form increased chromatin condensation which is a distinct characteristic of apoptotic cells. From FACS analysis, it is also clear that these drugs induce G2-M arrest and apoptosis in breast cancer cells. Further, transcript and protein level expression of MBDs and DNMTs is also affected - after treatment with epigenetic drugs; the level of transcripts/mRNA of MBDs and DNMTs has consistently increased in general. The increase in level of gene expression is substantiated at the protein level also where treated cells show higher expression of DNMT1, DNMT3A, DNMT3B, and MBD proteins in comparison to untreated cells. In case of tissue samples, the expression of different DNMTs is tissue stage-specific. DNMT1 exhibits significantly higher expression in the metastatic stage, whereas, DNMT3A and DNMT3B have higher expression in the primary stage in comparison to the metastatic samples. CONCLUSION: The epigenetic modulators AZA, TSA, SFN, and SAM may provide opportunities for cancer prevention by regulating the components of epigenetic gene-silencing machinery especially DNMTs and MBDs.
RESUMEN
Caveolin-1 (CAV1) is an integral part of plasma membrane protein playing a vital role in breast cancer initiation and progression. CAV1 acts both as a tumor suppressor as well as an oncogene, and its activity is thus highly dependent on cellular environment. Keeping this fact in mind, the recent work is designed to reveal the role of CAV1 in inhibiting cancer cell progression in presence of epigenetic modulators like 5-aza-2'-deoxycytidine (AZA), trichostatin A (TSA), S-adenosyl methionine (SAM) and sulforaphane (SFN). Forced expression of CAV1 by AZA, TSA, and SFN is correlated to induction of apoptosis and inhibition of cell migration in breast cancer. In breast cancer along with promoter DNA methylation, other epigenetic mechanisms are also involved in CAV1 expression. These observations clearly provide a new scenario regarding the role of CAV1 in cancer and as a possible therapeutic target in breast cancer.
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Caveolina 1/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Regiones Promotoras Genéticas , Adulto , Compuestos Aza/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Caveolina 1/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Epigénesis Genética/efectos de los fármacos , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Genes Supresores de Tumor , Humanos , Células MCF-7 , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The dynamic nature of chromatin and its myriad modifications play a crucial role in gene regulation (expression and repression) during development, cellular survival, homeostasis, ageing, and apoptosis/death. Histone 3 lysine 4 methylation (H3K4 methylation) catalyzed by H3K4 specific histone methyltransferases is one of the more critical chromatin modifications that is generally associated with gene activation. Additionally, the deposition of H3 variant(s) in conjunction with H3K4 methylation generates an intricately reliable epigenetic regulatory circuit that guides transcriptional activity in normal development and homeostasis. Consequently, alterations in this epigenetic circuit may trigger disease development. The mechanistic relationship between H3 variant deposition and H3K4 methylation during normal development has remained foggy. However, recent investigations in the field of chromatin dynamics in various model organisms, tumors, cancer tissues, and cell lines cultured without and with therapeutic agents, as well as from model reconstituted chromatins reveal that there may be different subsets of chromatin assemblage with specific patterns of histone replacement executing similar functions. In this light, we attempt to explain the intricate control system that maintains chromatin structure and dynamics during normal development as well as during tumor development and cancer progression in this review. Our focus is to highlight the contribution of H3K4 methylation-histone variant crosstalk in regulating chromatin architecture and subsequently its function.