ABSTRACT
Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.
Subject(s)
Actins , Calcium-Binding Proteins , Calcium , Wound Healing , Actins/metabolism , Animals , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Movement , Drosophila , Drosophila Proteins , Mice , Signal TransductionABSTRACT
Single cell multi-omics analysis has the potential to yield a comprehensive understanding of the cellular events that underlie the basis of human diseases. The cardinal feature to access this information is the technology used for single-cell isolation, barcoding, and sequencing. Most currently used single-cell RNA-sequencing platforms have limitations in several areas including cell selection, documentation and library chemistry. In this study, we describe a novel high-throughput, full-length, single-cell RNA-sequencing approach that combines the CellenONE isolation and sorting system with the ICELL8 processing instrument. This method offers substantial improvements in single cell selection, documentation and capturing rate. Moreover, it allows the use of flexible chemistry for library preparations and the analysis of living or fixed cells, whole cells independent of sizing and morphology, as well as of nuclei. We applied this method to dermal fibroblasts derived from six patients with different segmental progeria syndromes and defined phenotype associated pathway signatures with variant associated expression modifiers. These results validate the applicability of our method to highlight genotype-expression relationships for molecular phenotyping of individual cells derived from human patients.
Subject(s)
High-Throughput Nucleotide Sequencing , Single-Cell Analysis , Aging , Genotype , High-Throughput Nucleotide Sequencing/methods , Humans , Phenotype , RNA , Single-Cell Analysis/methodsABSTRACT
Posttranslational modifications play a crucial role in regulating gene expression. Among these modifications, arginine methylation has recently attracted tremendous attention due to its role in multiple cellular functions. This review discusses the recent advances that have established arginine methylation as a major player in determining cytoplasmic messenger RNA (mRNA) fate. We specifically focus on research that implicates arginine methylation in regulating mRNA translation, decay, and RNA granule dynamics. Based on this research, we highlight a few emerging future avenues that will lead to exciting discoveries in this field.
Subject(s)
Arginine/metabolism , Cytoplasm/metabolism , Protein Processing, Post-Translational , RNA, Messenger/metabolism , Gene Expression Regulation , Methylation , Protein Processing, Post-Translational/genetics , RNA, Messenger/geneticsABSTRACT
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.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/therapy , MicroRNAs/administration & dosage , Molecular Targeted Therapy/methods , Animals , Breast Neoplasms/genetics , Disease Management , Female , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/geneticsABSTRACT
Wnt signaling is crucial for proper development, tissue homeostasis and cell cycle regulation. A key role of Wnt signaling is the GSK3ß-mediated stabilization of ß-catenin, which mediates many of the critical roles of Wnt signaling. In addition, it was recently revealed that Wnt signaling can also act independently of ß-catenin. In fact, Wnt mediated stabilization of proteins (Wnt/STOP) that involves an LRP6-DVL-dependent signaling cascade is required for proper regulation of mitosis and for faithful chromosome segregation in human somatic cells. We show that inhibition of Wnt/LRP6 signaling causes whole chromosome missegregation and aneuploidy by triggering abnormally increased microtubule growth rates in mitotic spindles, and this is mediated by increased GSK3ß activity. We demonstrate that proper mitosis and maintenance of numerical chromosome stability requires continuous basal autocrine Wnt signaling that involves secretion of Wnts. Importantly, we identified Wnt10b as a Wnt ligand required for the maintenance of normal mitotic microtubule dynamics and for proper chromosome segregation. Thus, a self-maintaining Wnt10b-GSK3ß-driven cellular machinery ensures the proper execution of mitosis and karyotype stability in human somatic cells.
Subject(s)
Aneuploidy , Dishevelled Proteins/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Proto-Oncogene Proteins/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway/genetics , beta Catenin/metabolism , Chromosomal Instability/drug effects , Chromosomal Instability/genetics , Chromosome Segregation/drug effects , Chromosome Segregation/genetics , Gene Silencing , HCT116 Cells , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Microtubules/metabolism , Mitosis/drug effects , Mitosis/genetics , Protein Stability , Proto-Oncogene Proteins/genetics , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Spindle Apparatus/metabolism , Transfection , Wnt Proteins/genetics , Wnt Signaling Pathway/drug effectsABSTRACT
Scd6 is a conserved RGG-motif protein which represses translation by binding eIF4G through its RGG-motif. Lsm and FDF are two other conserved domains present in the protein, however the role of both these domains is unclear. We provide evidence in this report that the Lsm domain is important for the role of Scd6 in translation. Mutant of Scd6 lacking the Lsm domain does not cause overexpression growth defect in a manner comparable to the wild type. Similar results were observed with two distinct point mutants of Scd6 wherein putative RNA-binding motifs DxEKxTV and YVG were mutated. Upon overexpression, the three mutants were defective in inducing formation of P-bodies and stress granules which are conserved sites of translation repression. Importantly localization to granules in response to glucose deprivation and sodium azide stress was defective for Lsm domain mutants indicating that the inability to localize to granules could be a reason for their defective role in translation. Deletion of scd6 impairs Lsm1 foci formation upon glucose deprivation stress which could not be rescued by complementation with Lsm-domain deletion mutant of Scd6 when compared to the full-length protein. Put together, our results highlight the role of Lsm domain and its specific motifs in Scd6 activity and provide crucial insight into its function.
Subject(s)
Protein Biosynthesis , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cytoplasmic Granules/metabolism , Lipoproteins/genetics , Lipoproteins/metabolism , Mutation , Pheromones/genetics , Pheromones/metabolism , Protein Domains , RNA, Messenger/metabolism , Ribonucleoproteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Stress, PhysiologicalABSTRACT
The fate of messenger RNA in cytoplasm plays a crucial role in various cellular processes. However, the mechanisms that decide whether mRNA will be translated, degraded or stored remain unclear. Single stranded nucleic acid binding protein (Sbp1), an Arginine-Glycine-Glycine (RGG-motif) protein, is known to promote transition of mRNA into a repressed state by binding eukaryotic translation initiation factor 4G1 (eIF4G1) and to promote mRNA decapping, perhaps by modulation of Dcp1/2 activity. Sbp1 is known to be methylated on arginine residues in RGG-motif; however, the functional relevance of this modification in vivo remains unknown. Here, we report that Sbp1 is arginine-methylated in an hnRNP methyl transferase (Hmt1)-dependent manner and that methylation is enhanced upon glucose deprivation. Characterization of an arginine-methylation-defective (AMD) mutant provided evidence that methylation affects Sbp1 function in vivo. The AMD mutant is compromised in causing growth defect upon overexpression, and the mutant is defective in both localizing to and inducing granule formation. Importantly, the Sbp1-eIF4G1 interaction is compromised both for the AMD mutant and in the absence of Hmt1. Upon overexpression, wild-type Sbp1 increases localization of another RGG motif containing protein, Scd6 (suppressor of clathrin deficiency) to granules; however, this property of Sbp1 is compromised in the AMD mutant and in the absence of Hmt1, indicating that Sbp1 repression activity could involve other RGG-motif translation repressors. Additionally, the AMD mutant fails to increase localization of the decapping activator DEAD box helicase homolog to foci and fails to rescue the decapping defect of a dcp1-2Δski8 strain, highlighting the role of Sbp1 methylation in decapping. Taken together, these results suggest that arginine methylation modulates Sbp1 role in mRNA fate determination.
Subject(s)
Arginine/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Selenium-Binding Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Blotting, Western , Circular Dichroism , Cytoplasmic Granules/metabolism , Methylation , Protein Binding , Protein Processing, Post-Translational , RNA, Messenger/metabolismABSTRACT
Regulation of mRNA translation plays a key role in the control of gene expression. Scd6, a conserved RGG-motif containing protein represses translation by binding to translation initiation factor eIF4G1. Here we report that Scd6 binds itself in RGG-motif dependent manner and self-association regulates its repression activity. Scd6 self-interaction competes with eIF4G1 binding and methylation of Scd6 RGG-motif by Hmt1 negatively affects self-association. Results pertaining to Sbp1 indicate that self-association could be a general feature of RGG-motif containing translation repressor proteins. Taken together, our study reveals a mechanism of regulation of eIF4G-binding RGG-motif translation repressors.
Subject(s)
Eukaryotic Initiation Factor-4G/chemistry , Eukaryotic Initiation Factor-4G/metabolism , Protein Biosynthesis , Repressor Proteins/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Motifs , Arginine/metabolism , Methylation , Protein Binding , Protein Multimerization , Saccharomyces cerevisiae/metabolism , Stress, PhysiologicalABSTRACT
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.
Subject(s)
Cell Survival/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Inflammation/metabolism , Plant Extracts/pharmacology , Prostatic Neoplasms , Rubiaceae/chemistry , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Histone Deacetylase 1/genetics , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/genetics , Histone Deacetylase 2/metabolism , Humans , Inflammation/genetics , Male , Pentacyclic Triterpenes , Phytochemicals , Plant Extracts/chemistry , Plant Leaves/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , SitosterolsABSTRACT
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.
Subject(s)
Antigens, CD/genetics , Breast Neoplasms/genetics , Cadherins/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Gene Expression Regulation, Neoplastic , Gene Silencing , Snail Family Transcription Factors/genetics , Antioxidants/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Butadienes/pharmacology , Cadherins/deficiency , Cell Line , DNA (Cytosine-5-)-Methyltransferase 1/antagonists & inhibitors , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA Methylation , Epithelial-Mesenchymal Transition/drug effects , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Histone Deacetylase 1/genetics , Histone Deacetylase 1/metabolism , Histones/genetics , Histones/metabolism , Humans , Hydrogen Peroxide/antagonists & inhibitors , Hydrogen Peroxide/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Kaplan-Meier Estimate , MCF-7 Cells , Nitriles/pharmacology , Pentacyclic Triterpenes/pharmacology , Signal Transduction , Sitosterols/pharmacology , Snail Family Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , MicroRNAs/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Prostatic Neoplasms/genetics , 3' Untranslated Regions , Caveolin 1/genetics , Cell Line, Tumor , Cell Proliferation , Cell Survival , Chromatin/metabolism , Down-Regulation , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Humans , Male , Methylation , Prostatic Neoplasms/metabolismABSTRACT
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.
Subject(s)
DNA Methylation/genetics , Lung Neoplasms/genetics , Tumor Suppressor Proteins/genetics , A549 Cells , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, Tumor Suppressor/physiology , Humans , Microtubules/physiology , Promoter Regions, Genetic/geneticsABSTRACT
Infertility is a complex pathophysiological condition. It may caused by specific or multiple physical and physiological factors, including abnormalities in homeostasis, hormonal imbalances and genetic alterations. In recent times various studies implicated that, aberrant epigenetic mechanisms are associated with reproductive infertility. There might be transgenerational effects associated with epigenetic modifications of gametes and studies suggest the importance of alterations in epigenetic modification at early and late stages of gametogenesis. To determine the causes of infertility it is necessary to understand the altered epigenetic modifications of associated gene and mechanisms involved therein. This review is devoted to elucidate the recent mechanistic advances in regulation of genes by epigenetic modification and emphasizes their possible role related to reproductive infertility. It includes environmental, nutritional, hormonal and physiological factors and influence of internal structural architecture of chromatin nucleosomes affecting DNA and histone modifications in both male and female gametes, early embryogenesis and offspring. Finally, we would like to emphasize that research on human infertility by gene knock out of epigenetic modifiers genes must be relied upon animal models.
Subject(s)
Infertility/genetics , Animals , Environment , Epigenomics , Female , Humans , MaleABSTRACT
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.
Subject(s)
Breast Neoplasms/genetics , Cadherins/metabolism , Cell Movement/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation/genetics , Histones/metabolism , Lysine/metabolism , MicroRNAs/genetics , Antigens, CD , Base Sequence , Breast Neoplasms/pathology , Cell Line, Tumor , Chromatin/metabolism , DNA (Cytosine-5-)-Methyltransferase 1 , Disease Progression , Down-Regulation/drug effects , Epigenesis, Genetic/drug effects , Female , Fluorescent Antibody Technique , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/metabolism , Neoplasm Grading , Neoplasm Staging , Prognosis , Promoter Regions, Genetic/genetics , RNA, Small Interfering/metabolism , Transfection , Wound Healing/geneticsABSTRACT
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.
Subject(s)
Breast Neoplasms/drug therapy , Curcumin/pharmacology , DNA-Binding Proteins/physiology , Oncogene Proteins/physiology , Receptors, Estrogen/analysis , Apoptosis/drug effects , Breast Neoplasms/chemistry , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Movement , Cell Proliferation , DNA-Binding Proteins/genetics , Female , Humans , Jumonji Domain-Containing Histone Demethylases/analysis , MCF-7 Cells , Molecular Chaperones , Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt/physiology , Proto-Oncogene Proteins c-bcl-2/analysis , RNA-Binding ProteinsABSTRACT
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.
Subject(s)
Benzoquinones/chemistry , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylases/chemistry , Amino Acid Sequence , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Benzoquinones/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Damage/drug effects , Enzyme Activation/drug effects , Female , Gene Expression , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Humans , Hydrogen Bonding , Models, Molecular , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Sequence Data , Protein Binding , Sequence Alignment , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Clusterin/genetics , Colonic Neoplasms/genetics , Histones/metabolism , Adult , Aged , Base Sequence , Cell Death/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Clusterin/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , DNA Methylation , Gene Expression Regulation, Neoplastic , Humans , Middle Aged , Molecular Sequence Data , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Processing, Post-Translational/physiology , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Biflavonoids/pharmacology , Catechin/pharmacology , DNA Modification Methylases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Proanthocyanidins/pharmacology , Amino Acid Sequence , Animals , Breast/drug effects , Breast/metabolism , Breast/pathology , Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Catechin/analogs & derivatives , Cell Line, Tumor , DNA Methylation/drug effects , DNA Modification Methylases/chemistry , DNA Modification Methylases/metabolism , Female , Humans , Mice , Molecular Docking Simulation , Molecular Sequence Data , Sequence AlignmentABSTRACT
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.
ABSTRACT
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.