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P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.
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G-Cuádruplex , Proteína Quinasa 12 Activada por Mitógenos , Neoplasias de la Mama Triple Negativas , Humanos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Neoplasias de la Mama Triple Negativas/genética , Proteína Quinasa 12 Activada por Mitógenos/genéticaRESUMEN
Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.
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G-Cuádruplex , Neoplasias , Fotoquimioterapia , Animales , ADN/metabolismo , Daño del ADN , Replicación del ADN , Inestabilidad Genómica , Neoplasias/genética , Neoplasias/terapia , Estrés Oxidativo , Fármacos Fotosensibilizantes/farmacología , Pez Cebra/genética , Pez Cebra/metabolismo , Fotoquimioterapia/métodosRESUMEN
G-Quadruplex (GQ) and i-motif structures are the paradigmatic examples of nonclassical tetrastranded nucleic acids having multifarious biological functions and widespread applications in therapeutics and material science. Recently, tetraplexes emerged as promising anticancer targets due to their structural robustness, gene-regulatory roles, and predominant distribution at specific loci of oncogenes. However, it is arguable whether the i-motif evolves in the complementary single-stranded region after GQ formation in its opposite strand and vice versa. In this review, we address the prerequisites and significance of the simultaneous and/or mutually exclusive formation of GQ and i-motif structures at complementary and sequential positions in duplexes in the cellular milieu. We discussed how their dynamic interplay Sets up cellular homeostasis and exacerbates carcinogenesis. The review gives insights into the spatiotemporal formation of GQ and i-motifs that could be harnessed to design different types of reporter systems and diagnostic platforms for potential bioanalytical and therapeutic intervention.
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G-Cuádruplex , Genoma Humano , Daño del ADN , Humanos , Plasticidad Neuronal , Conformación de Ácido Nucleico , Especies Reactivas de Oxígeno/química , Telómero/genéticaRESUMEN
F-box protein 31 (FBXO31) is a reported putative tumor suppressor, and its inactivation due to loss of heterozygosity is associated with cancers of different origins. An emerging body of literature has documented FBXO31's role in preserving genome integrity following DNA damage and in the cell cycle. However, knowledge regarding the role of FBXO31 during normal cell-cycle progression is restricted to its functions during the G2/M phase. Interestingly, FBXO31 levels remain high even during the early G1 phase, a crucial stage for preparing the cells for DNA replication. Therefore, we sought to investigate the functions of FBXO31 during the G1 phase of the cell cycle. Here, using flow cytometric, biochemical, and immunofluorescence techniques, we show that FBXO31 is essential for maintaining optimum expression of the cell-cycle protein cyclin A for efficient cell-cycle progression. Stable FBXO31 knockdown led to atypical accumulation of cyclin A during the G1 phase, driving premature DNA replication and compromised loading of the minichromosome maintenance complex, resulting in replication from fewer origins and DNA double-strand breaks. Because of these inherent defects in replication, FBXO31-knockdown cells were hypersensitive to replication stress-inducing agents and displayed pronounced genomic instability. Upon entering mitosis, the cells defective in DNA replication exhibited a delay in the prometaphase-to-metaphase transition and anaphase defects such as lagging and bridging chromosomes. In conclusion, our findings establish that FBXO31 plays a pivotal role in preserving genomic integrity by maintaining low cyclin A levels during the G1 phase for faithful genome duplication and segregation.
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Ciclina A/metabolismo , Replicación del ADN/genética , Proteínas F-Box/metabolismo , Genoma Humano/genética , Proteínas Supresoras de Tumor/metabolismo , Ciclo Celular/genética , Cromatina/genética , Proteínas F-Box/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Cinética , Células MCF-7 , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Ubiquitinación/genéticaRESUMEN
c-MYC proto-oncogene harbours a transcription-inhibitory quadruplex-forming scaffold (Pu27) upstream P1 promoter providing anti-neoplastic therapeutic target. Previous reports showed the binding profile of human Cathelicidin peptide (LL37) and telomeric G-quadruplex. Here, we truncated the quadruplex-binding domain of LL37 to prepare a small library of peptides through site-specific amino acid substitution. We investigated the intracellular selectivity of peptides for Pu27 over other oncogenic quadruplexes and their role in c-MYC promoter repression by dual-luciferase assays. We analysed their thermodynamics of binding reactions with c-MYC quadruplex isomers (Pu27, Myc22, Pu19) by Isothermal Titration Calorimetry. We discussed how amino acid substitutions and peptide helicity enhanced/weakened their affinities for c-MYC quadruplexes and characterized specific non-covalent inter-residual interactions determining their selectivity. Solution NMR structure indicated that KR12C, the best peptide candidate, selectively stabilized the 5'-propeller loop of c-MYC quadruplex by arginine-driven electrostatic-interactions at the sugar-phosphate backbone while KR12A peptide destabilized the quadruplex inducing a single-stranded hairpin-like conformation. Chromatin immunoprecipitations envisaged that KR12C and KR12A depleted and enriched Sp1 and NM23-H2 (Nucleoside diphosphate kinase) occupancy at Pu27 respectively supporting their regulation in stabilizing and unfolding c-MYC quadruplex in MCF-7 cells. We deciphered that selective arresting of c-MYC transcription by KR12C triggered apoptotic-signalling pathway via VEGF-A-BCL-2 axis.
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Péptidos Catiónicos Antimicrobianos/farmacología , Apoptosis/efectos de los fármacos , G-Cuádruplex/efectos de los fármacos , Neoplasias/patología , Conformación de Ácido Nucleico/efectos de los fármacos , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-myc/efectos de los fármacos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Ensayos de Selección de Medicamentos Antitumorales , Genes myc/efectos de los fármacos , Humanos , Células MCF-7 , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genética , CatelicidinasRESUMEN
The nuclease hypersensitive element III1 (NHE III1) upstream c-MYC promoter harbors a transcription-silencing G-quadruplex (Pu27) element. Dynamic turnover of various transcription factors (TFs) across Pu27 to control c-MYC transcription homeostasis is enigmatic. Here, we reveal that native Pu27 evolves truncated G-quadruplex isomers (Pu19, Pu22, Pu24, and Pu25) in cells that are optimal intracellular targets of specific TFs in a sequence- and structure-dependent manner. Nuclear magnetic resonance and isothermal titration calorimetry envisaged that NM23-H2 (nucleoside diphosphate kinase) and nucleolin induce conformational fluctuations in Pu27 to sample specific conformationally restricted conformer(s). Structural investigations revealed that the flanking guanines at 5'-Pu27 control solvent exposure at G-quartets upon NM23-H2 and nucleolin binding driving Pu27 unfolding and folding, respectively. Transient chromatin immunoprecipitations confirmed that NM23-H2 drives the conformation switch to Pu24 that outcompetes nucleolin recruitment. Similarly, nucleolin arrests Pu27 in the Pu22 conformer minimizing NM23-H2 binding at Pu27. hnRNPK (heterogeneous nuclear ribonucleoprotein K) positively regulates NM23-H2 and nucleolin association at Pu27 despite their antagonism. On the basis of these results, we simulated the transcription kinetics in a feed-forward loop in which the transcription output responds to hnRNPK-induced early activation via NM23-H2 association, which favors Pu24 formation at NHE III1 reducing nucleolin occupancy and driving quadruplex unfolding to initiate transcription. NM23-H2 further promotes hnRNPK deposition across NHE III1 altering Pu27 plasticity that finally enriches the nucleolin abundance to drive Pu22 formation and weaken NM23-H2 binding to extinguish transcription. This mechanism involves three positive feedback loops (NM23-H2-hnRNPK, NM23-H2-CNBP, and hnRNPK-nucleolin) and one negative feedback loop (NM23-H2-nucleolin) controlling optimal turnover and residence time of TFs at Pu27 to homeostatically regulate c-MYC transcription.
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ADN/química , G-Cuádruplex , Homeostasis , Proteínas Proto-Oncogénicas c-myc/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/química , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Isomerismo , Nucleósido Difosfato Quinasas NM23/química , Nucleósido Difosfato Quinasas NM23/genética , Nucleósido Difosfato Quinasas NM23/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , NucleolinaRESUMEN
Identification of key amino acids is required for development of efficient cell-penetrating peptides (CPPs) and has tremendous implications in medicine. Extensive research work has enlightened us about the importance of two amino acids, arginine and tryptophan, in cell penetration. Here, we present a top-down approach to show how spatial positions of two tryptophans regulate the cellular entry and nuclear localization. This enables us to develop short, non-toxic tetrapeptides with excellent potential for cell penetration and nuclear localization. Among them, Glu-Thr-Trp-Trp (ETWW) emerges as the most promising. Results suggest that it enters into cancer cells following an endocytic pathway and binds at the major groove of nuclear DNA, where successive tryptophan plays major role. We subsequently show that it is not a P-glycoprotein substrate and is non-toxic to PC12-derived neurons, suggesting its excellent potential as a CPP. Furthermore, its potential as a CPP is validated in multi-cellular 3D cell culture (spheroid) and in in vivo mice model. This study provides major fundamental insights about the positional importance of tryptophan and opens new avenues toward the development of next-generation CPPs and major-groove-specific anticancer drugs.
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Núcleo Celular/metabolismo , Péptidos de Penetración Celular/metabolismo , Triptófano/metabolismo , Animales , Núcleo Celular/química , Péptidos de Penetración Celular/química , Células Cultivadas , Humanos , Células MCF-7 , Ratones , Células PC12 , Ratas , Triptófano/químicaRESUMEN
Genomic DNA exhibits an innate ability to manifest diverse sequence-dependent secondary structures, serving crucial functions in gene regulation and cellular equilibrium. While extensive research has confirmed the formation of G-quadruplex structures by guanine-rich sequences in vitro and in cells, recent investigations have turned the quadruplex community's attention to the cytosine (C)-rich complementary strands that can adopt unique tetra-stranded conformation, termed as intercalated motif or i-motif. I-motifs are stabilized by hemi-protonated C:CH+ base pairs under acidic conditions. Initially, the in vivo occurrence of i-motifs was underestimated because their formation is favored at non-physiological pH. However, groundbreaking research utilizing the structure-specific iMab antibody and high-throughput sequencing have recently detected their conserved dispersion throughout the genome, challenging previous assumptions. Given the evolving nature of this research field, it becomes imperative to conduct independent in vitro experiments aimed at identifying potential i-motif formation in C-rich sequences and consolidating the findings to address the properties of i-motifs. This chapter serves as an introductory guide for the swift identification of novel i-motifs, where we present an experimental framework for investigating and characterizing i-motif sequences in vitro. In this chapter, we selected a synthetic oligonucleotide (C7T3) sequence and outlined appropriate methodologies for annealing the i-motif structure into suitable buffers. Then, we validated its formation by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopy. Finally, we provided a thorough account of the step-by-step procedures to investigate the effect of i-motif formation on the stalling or retardation of DNA replication using high resolution primer extension assays.
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ADN , G-Cuádruplex , Conformación de Ácido Nucleico , ADN/química , Emparejamiento Base , Espectroscopía de Resonancia Magnética , Dicroismo CircularRESUMEN
We have elucidated the hnRNP K promoter as a hotspot for tetraplex-based molecular switches receptive to micro-environmental stimuli. We have characterised the structural features of four tetraplex-forming loci and identified them as binding sites of transcription factors. These segments form either G-quadruplex or i-motif structures, the structural dynamicity of which has been studied in depth via several biophysical techniques. The tetraplexes display high dynamicity and are influenced by both pH and KCl concentrations in vitro. The loci complementary to these sequences form additional non-canonical secondary structures. In the cellular context, the most eminent observation of this study is the binding of hnRNP K to the i-motif forming sequences in its own promoter. We are the first to report a probable transcriptional autoregulatory function of hnRNP K in coordination with higher-order DNA structures. Herein, we also report the positive interaction of the endogenous tetraplexes with Sp1, a well-known transcriptional regulator. Treatment with tetraplex-specific small molecule ligands further uncovered G-quadruplexes' functioning as repressors and i-motifs as activators in this context. Together, our findings strongly indicate the critical regulatory role of the identified tetraplex elements in the hnRNP K promoter.Communicated by Ramaswamy H. Sarma.
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LncRNAs and miRNAs, being the master regulators of gene expression, are crucial functional mediators in cancer. Our study unveils the critical regulatory role of the metastatic long non-coding RNA LINC00273 as the master regulator of oncogenes involved in cancer metastasis, stemness, and chemoresistance via its miRNA sponging mechanism. M2 (a salt of bis-Schiff base) mediated G quadruplex (G4) stabilization at the LINC00273 gene promoter remarkably inhibits LINC00273 transcription. Therefore, low-level LINC00273 transcripts are unable to efficiently sponge the miRNAs, which subsequently become available to bind and downregulate their target oncogenes. We have observed significantly different global transcriptomic scenarios in LINC00273 upregulated and downregulated circumstances in MDA-MB-231 triple-negative breast cancer model. Additionally, we have found the G4 sequence in the LINC00273 RNA to play a critical role in miRNA sequestration. miRNAs (miR-6789-5p, miR200b, miR-125b-5p, miR-4268, miR3978) have base pairing complementarity within the G4 region of LINC00273 RNA and the 3'-UTR (untranslated region) of MAPK12, TGF-ß1, and SIX-1 transcripts. We have reported TGF-ß1, SIX-1, and MAPK12 to be the direct downstream targets of LINC00273. The correlation between abnormal expression of lncRNA LINC00273 and TNBC aggressiveness strongly evidenced in our study shall accelerate the development of lncRNA-based anti-metastatic therapeutics.
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Regulación Neoplásica de la Expresión Génica , MicroARNs , Metástasis de la Neoplasia , ARN Largo no Codificante , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , MicroARNs/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , ARN Largo no Codificante/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
G-Quadruplex (GQ) nucleic acids are promising therapeutic targets in anticancer research due to their structural robustness, polymorphism, and gene-regulatory functions. Here, we presented the structure-activity relationship of carbazole-based monocyanine ligands using region-specific functionalization with benzothiazole (TCA and TCZ), lepidine (LCA and LCZ), and quinaldine (QCA and QCZ) acceptor moieties and evaluated their binding profiles with different oncogenic GQs. Their differential turn-on fluorescence emission upon GQ binding confirmed the GQ-to-duplex selectivity of all carbazole ligands, while the isothermal titration calorimetry results showed selective interactions of TCZ and TCA to c-MYC and BCL-2 GQs, respectively. The aldehyde group in TCA favors stacking interactions with the tetrad of BCL-2 GQ, whereas TCZ provides selective groove interactions with c-MYC GQ. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) showed that these molecules interfere with the recruitment of specific transcription factors at c-MYC and BCL-2 promoters and stabilize the promoter GQ structures to inhibit their constitutive transcription in cancer cells. Their intrinsic turn-on fluorescence response with longer lifetimes upon GQ binding allowed real-time visualization of GQ structures at subcellular compartments. Confocal microscopy revealed the uptake of these ligands in the nucleoli, resulting in nucleolar stress. ChIP studies further confirmed the inhibition of Nucleolin occupancy at multiple GQ-enriched regions of ribosomal DNA (rDNA) promoters, which arrested rRNA biogenesis. Therefore, carbazole ligands act as the "double-edged swords" to arrest c-MYC and BCL-2 overexpression as well as rRNA biogenesis, triggering synergistic inhibition of multiple oncogenic pathways and apoptosis in cancer cells.
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BACKGROUND: Aberrant expression of Zinc-finger E-box binding homeobox 1 (ZEB1), which remains repressed in normal cells, is frequently associated with cancer aggressiveness. However, transcriptional mechanism underlying such atypical ZEB1 expression in cancer is not yet well-understood. METHODS: ZEB1 promoter G-quadruplexes were studied and modeled extensively using circular dichroism, fluorescence spectroscopy, ITC and DMS protection assay. Luciferase assay, qPCR, FAIRE, ChIP, western blotting, confocal microscopy was used to access the regulation of ZEB1 transcription. RESULTS: Our study unravels the occupancy of nucleolin to ZEB1 promoter as a crucial determinant which facilitates the binding of SP1 transcription factor to chromatin, by locally remodelling the region. SP1, subsequently, recruits P300 acetyl transferase leading to enriched acetyl-histone H3 at promoter and activates ZEB1 transcription. ZEB1 promoter analysis identifies presence of four putative G-quadruplex (G4) forming motifs within 700 bp of TSS; each quadruplex is characterized structurally in details with an array of biophysical techniques. Surprisingly, stabilization of G4 with cationic porphyrin TMPyP4 represses its transcription and eventually impedes cell invasiveness. CONCLUSIONS: TMPyP4 binding to a selected G4 motif (5' -534/-511-3' from TSS), where nucleolin/SP1/P300 co-occupies, prevents the association of nucleolin which consequently hinders SP1 binding, leading to chromatin compactness and transcriptional repression. GENERAL SIGNIFICANCE: Our findings demonstrate an epigenetic mechanism of ZEB1 reactivation where dynamic occupancy of transcription regulators encompassing a G4 motif is crucial and thus, small molecule induced G-quadruplex stabilization may act as a potential molecular switch to turn-off gene expression.
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Reprogramación Celular , Epigénesis Genética , G-Cuádruplex , Regulación Neoplásica de la Expresión Génica , Neoplasias/patología , Regiones Promotoras Genéticas , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Apoptosis , Proliferación Celular , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Células Tumorales Cultivadas , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
BACKGROUND: The transcription-inhibitory G-Quadruplex(Pu27-GQ) at c-MYC promoter is challenging to target due to structural heterogeneity. Nucleoside diphosphate kinase (NM23-H2) specifically binds and unfolds Pu27-GQ to increase c-MYC transcription. Here, we used Inosine 5'-diphosphate (IDP) to disrupt NM23-H2-Pu27-GQ interactions and arrest c-MYC transcription without compromising NM23-H2-mediated kinase properties. METHODS: Site-directed mutagenesis,31P-NMR and STD-NMR studies delineate the epitope of NM23-H2-IDP complex and characterize specific amino acids in NM23-H2 involved in Pu27-GQ and IDP interactions. Immunoprecipitations and phosphohistidine-immunoblots reveal how IDP blocks NM23-H2-Pu27 association to downregulate c-MYC transcription in MDAMB-231 cells exempting NM23-H2-mediated kinase properties. RESULTS: NMR studies show that IDP binds to the Guanosine diphosphate-binding pocket of NM23-H2 (KD = 5.0 ± 0.276 µM). Arg88-driven hydrogen bonds to the terminal phosphate of IDP restricts P-O-P bond-rotation increasing its pKa (∆pKa = 0.85 ± 0.0025).9-inosinyl moiety of IDP is stacked over Phe60 phenyl ring driving trans-conformation of inosine and axial geometry of pyrophosphates. Chromatin immunoprecipitations revealed that these interactions rescue NM23-H2-driven Pu27-GQ unfolding, which triggers Nucleolin recruitment and lowers Sp1 occupancy at c-MYC promoter stabilizing Pu27-GQ. This silences c-MYC transcription that reduces c-MYC-Sp1 association amplifying Sp1 recruitment across P21 promoter stimulating P21 transcription and G2/M arrest. CONCLUSIONS: IDP synergizes the effects of Pu27-GQ-interacting compounds to abrogate c-MYC transcription and induce apoptosis in MDAMB-231 cells by disrupting NM23-H2-Pu27-GQ interactions without affecting NM23-H2-mediated kinase properties. GENERAL SIGNIFICANCE: Our study provides a pragmatic approach for developing NM23-H2-targeting regulators to rescue NM23-H2 binding at structurally ambiguous Pu27-GQ that synergizes the anti-tumorigenic effects of GQ-based therapeutics with minimized off-target effects.
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G-Cuádruplex , Inosina Difosfato/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Línea Celular Tumoral , Puntos de Control de la Fase G2 del Ciclo Celular , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Modelos Moleculares , Nucleósido-Difosfato Quinasa/química , Regiones Promotoras Genéticas/genética , Conformación Proteica , Transcripción GenéticaRESUMEN
We designed novel 4'-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in ≈4.9-fold more efficient knockdown than unmodified siRNA.
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Guanidina/química , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Guanidina/análogos & derivados , Modelos Moleculares , ARN Interferente Pequeño/genética , Ribonucleasas/sangre , Ribonucleasas/metabolismo , TermodinámicaRESUMEN
Correction for '4'-Guanidinium-modified siRNA: a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading' by Ganesh N. Nawale et al., Chem. Commun., 2019, 55, 9112-9115.
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Our environment is full of statistical regularities, and we are attuned to learn about these regularities by employing Statistical Learning (SL), a domain-general ability that enables the implicit detection of probabilistic regularities in our surrounding environment. The role of brain connectivity on SL has been previously explored, highlighting the relevance of structural and functional connections between frontal, parietal, and temporal cortices. However, whether SL can induce changes in the functional connections of the resting state brain has yet to be investigated. To address this question, we applied a pre-post design where participants (nâ¯=â¯38) were submitted to resting-state fMRI acquisition before and after in-scanner exposure to either an artificial language stream (formed by 4 concatenated words) or a random audio stream. Our results showed that exposure to an artificial language stream significantly changed (corrected pâ¯<â¯0.05) the functional connectivity between Right Posterior Cingulum and Left Superior Parietal Lobule. This suggests that functional connectivity between brain networks supporting attentional and working memory processes may play an important role in statistical learning.
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Encéfalo/fisiología , Lenguaje , Aprendizaje/fisiología , Percepción del Habla/fisiología , Estimulación Acústica , Adulto , Mapeo Encefálico , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Vías Nerviosas/fisiología , Estadística como Asunto , Adulto JovenRESUMEN
Reduced expression of Scaffold/Matrix Attachment Region Binding Protein 1 (SMAR1) is associated with various cancers resulting in poor prognosis of the diseases. However, the precise underlying mechanism elucidating the loss of SMAR1 requires ongoing study. Here, we show that SMAR1 is highly downregulated during aberrant Wnt3a signaling due to proteasomal degradation and predicted poor prognosis of colorectal cancer. However, substitution mutation (Arginine and Lysine to Alanine) in the D-box elements of SMAR1 viz. "RCHL" and "RQRL" completely abrogated its proteasomal degradation despite Wnt3a activity. SMAR1 inhibited Wnt/ß-catenin signaling by recruiting Histone deacetylase-5 to ß-catenin promoter resulting in reduced cell migration and invasion. Consequently, reduced tumor sizes in in-vivo NOD-SCID mice were observed that strongly associated with suppression of ß-catenin. However, loss of SMAR1 led to enriched H3K9 Acetylation in the ß-catenin promoter that further increased Wnt/ß-catenin signaling activities and enhanced colorectal cancer progression drastically. Using docking and isothermal titration calorimetric studies we show that small microbial peptides viz. AT-01C and AT-01D derived from Mycobacterium tuberculosis mask the D-box elements of SMAR1. These peptides stabilized SMAR1 expression that further inhibited metastatic SW480 colorectal cancer cell migration and invasion. Drastically reduced subcutaneous tumors were observed in in-vivo NOD-SCID mice upon administration of these peptides (25 mg/kg body weight) intraperitoneally. Taken together our structural studies, in-vitro and in-vivo results strongly suggest that the D-box elements of SMAR1 represent novel druggable targets, where the microbial peptides hold promise as novel colorectal cancer therapeutics.
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Recently, therapeutic implications of BCL-2 quadruplex invigorated the field of clinical oncology. This Keynote review discusses how a BCL-2 quadruplex-selective approach circumvents the limitations of existing therapeutics; and which improvisations might ameliorate the recent trends of quadruplex-based treatment.
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G-Cuádruplex , Genes bcl-2 , Neoplasias/tratamiento farmacológico , Neoplasias/genética , HumanosRESUMEN
Overexpression of c-MYC oncogene is associated with cancer pathology. Expression of c-MYC is regulated by the G-quadruplex structure formed in the G-rich segment of nuclease hypersensitive element (NHE III1), that is, "Pu27", which is localized in the promoter region. Ligand-induced stabilization of the Pu27 structure has been identified as a novel target for cancer therapeutics. Here, we have explored the library of synthetic compounds against the predefined binding site of Pu27. Three compounds were selected based on the docking analyses; they were further scrutinized using all atom molecular dynamics simulations in an explicit water model. Simulated trajectories were scrutinized for conformational stability and ligand binding free energy estimation; essential dynamic behavior was determined using principal component analysis. One of the molecules, "TPP (1-(3-(4-(1,2,3-thiadiazol-4-yl)phenoxy)-2-hydroxypropyl)-4-carbamoylpiperidinium)", with the best results was considered for further evaluation. The theoretical observations are supported well by biophysical analysis using circular dichroism, isothermal titration calorimetry, and high-resolution NMR spectroscopy indicating association of TPP with Pu27. The in vitro studies were then translated into c-MYC overexpression in the T47D breast cancer cell line. Biological evaluation through the MTT assay, flow cytometric assay, RT-PCR, and reporter luciferase assay suggests that TPP downregulates the expression of c-MYC oncogene by arresting its promoter region. In silico and in vitro observations cumulatively suggest that the novel skeleton of TPP could be a potential anticancer agent by stabilizing the G-quadruplex formed in the Pu27 and consequently downregulating the expression of c-MYC oncogene. Derivation of new molecules on its skeleton may confer anticancer therapeutics for the next generation.
RESUMEN
A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.