RESUMEN
Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.
Asunto(s)
Antineoplásicos , Péptidos de Penetración Celular , G-Cuádruplex , G-Cuádruplex/efectos de los fármacos , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Línea Celular Tumoral , Péptidos/química , Péptidos/farmacología , Ligandos , ADN/química , ADN/metabolismo , Complejo Shelterina/metabolismo , Complejo Shelterina/química , Unión ProteicaRESUMEN
Aiming to identify highly effective and selective G-quadruplex ligands as anticancer candidates, five natural compounds were investigated here, i.e., the alkaloids Canadine, D-Glaucine and Dicentrine, as well as the flavonoids Deguelin and Millettone, selected as analogs of compounds previously identified as promising G-quadruplex-targeting ligands. A preliminary screening with the G-quadruplex on the Controlled Pore Glass assay proved that, among the investigated compounds, Dicentrine is the most effective ligand of telomeric and oncogenic G-quadruplexes, also showing good G-quadruplex vs. duplex selectivity. In-depth studies in solution demonstrated the ability of Dicentrine to thermally stabilize telomeric and oncogenic G-quadruplexes without affecting the control duplex. Interestingly, it showed higher affinity for the investigated G-quadruplex structures over the control duplex (Kb~106 vs. 105 M-1), with some preference for the telomeric over the oncogenic G-quadruplex model. Molecular dynamics simulations indicated that Dicentrine preferentially binds the G-quadruplex groove or the outer G-tetrad for the telomeric and oncogenic G-quadruplexes, respectively. Finally, biological assays proved that Dicentrine is highly effective in promoting potent and selective anticancer activity by inducing cell cycle arrest through apoptosis, preferentially targeting G-quadruplex structures localized at telomeres. Taken together, these data validate Dicentrine as a putative anticancer candidate drug selectively targeting cancer-related G-quadruplex structures.
Asunto(s)
Antineoplásicos , G-Cuádruplex , Neoplasias , Humanos , Ligandos , Simulación de Dinámica Molecular , Antineoplásicos/farmacología , Telómero/metabolismoRESUMEN
HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.
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G-Cuádruplex , Proteína HMGB1/genética , Conformación de Ácido Nucleico , Telómero/genética , ADN/química , ADN/genética , Escherichia coli/genética , Proteína HMGB1/química , Humanos , Telomerasa/química , Telomerasa/genética , Telómero/químicaRESUMEN
Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Compuestos Azo/química , ADN/metabolismo , G-Cuádruplex , Osteosarcoma/tratamiento farmacológico , Piridinas/química , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , ADN/química , Humanos , Osteosarcoma/patología , Células Tumorales CultivadasRESUMEN
A focused library of newly designed monomeric and dimeric naphthalene diimides (NDIs) was analyzed in its ability to recognize specific G-quadruplex (G4) structures discriminating duplex DNA. The best G4 ligands-according to an affinity chromatography-based screening method named G4-CPG-were tested on human cancer and healthy cells, inducing DNA damage at telomeres, and in parallel, showing selective antiproliferative activity on HeLa cancer cells with IC50 values in the low nanomolar range. CD and fluorescence spectroscopy studies allowed detailed investigation of the interaction in solution with different G4 and duplex DNA models of the most promising NDI of the series, as determined by combining the biophysical and biological assays' data.
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Antineoplásicos/química , G-Cuádruplex/efectos de los fármacos , Iminas/química , Naftalenos/química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Daño del ADN , Células HeLa , Humanos , Iminas/farmacología , Ligandos , Naftalenos/farmacología , Telómero/efectos de los fármacosRESUMEN
The transcription factor CCCTC-binding factor (CTCF) modulates pleiotropic functions mostly related to gene expression regulation. The role of CTCF in large scale genome organization is also well established. A unifying model to explain relationships among many CTCF-mediated activities involves direct or indirect interactions with numerous protein cofactors recruited to specific binding sites. The co-association of CTCF with other architectural proteins such as cohesin, chromodomain helicases, and BRG1, further supports the interplay between master regulators of mammalian genome folding. Here, we report a comprehensive LC-MS/MS mapping of the components of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex co-associated with CTCF including subunits belonging to the core, signature, and ATPase modules. We further show that the localization patterns of representative SWI/SNF members significantly overlap with CTCF sites on transcriptionally active chromatin regions. Moreover, we provide evidence of a direct binding of the BRK-BRG1 domain to the zinc finger motifs 4-8 of CTCF, thus, suggesting that these domains mediate the interaction of CTCF with the SWI/SNF complex. These findings provide an updated view of the cooperative nature between CTCF and the SWI/SNF ATP-dependent chromatin remodeling complexes, an important step for understanding how these architectural proteins collaborate to shape the genome.
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Factor de Unión a CCCTC/genética , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Dedos de Zinc/genética , Adenosina Trifosfatasas/genética , Sitios de Unión/genética , Proteínas de Ciclo Celular/genética , Regulación de la Expresión Génica/genética , Genoma Humano/genética , Humanos , Complejos Multiproteicos/genética , Mapas de Interacción de Proteínas/genética , Espectrometría de Masas en Tándem , CohesinasRESUMEN
Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.
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Daño del ADN , Sirtuinas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Acetilación , Antineoplásicos Fitogénicos/farmacología , Camptotecina/farmacología , Línea Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Humanos , Inmunohistoquímica , Modelos Moleculares , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Proteolisis/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Sirtuinas/química , Especificidad por Sustrato , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/genética , UbiquitinaciónRESUMEN
Here, with the aim of obtaining insight into the intriguing selectivity of G-quadruplex (G4) ligands toward cancer compared to normal cells, a genetically controlled system of progressive transformation in human BJ fibroblasts was analyzed. Among the different comparative evaluations, we found a progressive increase of DNA damage response (DDR) markers throughout the genome from normal toward immortalized and transformed cells. More interestingly, sensitivity to G4 ligands strongly correlated with the presence of a basal level of DNA damage, including at the telomeres, where the chromosome ends were exposed to the DDR without concurrent induction of DNA repair activity, as revealed by the lack of 53BP1 recruitment and telomere aberrations. The link between telomere uncapping and the response to G4 stabilization was directly assessed by showing that a partial TRF2 depletion, causing a basal level of telomere localized DDR, rendered telomerized fibroblasts prone to G4-induced telomere damage and anti-proliferative defects. Taken together these data strongly indicate that the presence of a basal level of telomere-associated DDR is a determinant of susceptibility to G4 stabilization.
Asunto(s)
Daño del ADN , G-Cuádruplex/efectos de los fármacos , Neoplasias/genética , Telómero , Western Blotting , Inmunoprecipitación de Cromatina , Humanos , Hibridación Fluorescente in Situ , Células Tumorales CultivadasRESUMEN
Tumor angiogenesis is mainly mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic factor produced by cancer cells and active on the endothelium through the VEGF receptor 2 (VEGFR-2). Here we identify a G-rich sequence within the proximal promoter region of vegfr-2, able to form an antiparallel G-quadruplex (G4) structure. This G4 structure can be efficiently stabilized by small molecules with the consequent inhibition of vegfr-2 expression. Functionally, the G4-mediated reduction of VEGFR-2 protein causes a switching off of signaling components that, converging on actin cytoskeleton, regulate the cellular events leading to endothelial cell proliferation, migration and differentiation. As a result of endothelial cell function impairment, angiogenic process is strongly inhibited by G4 ligands both in vitro and in vivo. Interestingly, the G4-mediated antiangiogenic effect seems to recapitulate that observed by using a specific interference RNA against vegfr-2, and it is strongly antagonized by overexpressing the vegfr-2 gene. In conclusion, we describe the evidence for the existence of G4 in the promoter of vegfr-2, whose expression and function can be markedly inhibited by G4 ligands, thereby revealing a new, and so far undescribed, way to block VEGFR-2 as target for anticancer therapy.
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G-Cuádruplex , Neoplasias/irrigación sanguínea , Neovascularización Patológica , Regiones Promotoras Genéticas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Línea Celular Tumoral , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neovascularización Fisiológica , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
G-quadruplexes (G4s) are guanine-rich non-canonical secondary structures of nucleic acids that were identified in vitro almost half a century ago. Starting from the early 1980s, these structures were also observed in eukaryotic cells, first at the telomeric level and later in regulatory regions of cancer-related genes, in regulatory RNAs and within specific cell compartments such as lysosomes, mitochondria, and ribosomes. Because of the involvement of these structures in a large number of biological processes and in the pathogenesis of several diseases, including cancer, the interest in G4 targeting has exponentially increased in the last few years, and a great number of novel G4 ligands have been developed. Notably, G4 ligands represent a large family of heterogeneous molecules that can exert their functions by recognizing, binding, and stabilizing G4 structures in multiple ways. Regarding anti-cancer activity, the efficacy of G4 ligands was originally attributed to the capability of these molecules to inhibit the activity of telomerase, an enzyme that elongates telomeres and promotes endless replication in cancer cells. Thereafter, novel mechanisms through which G4 ligands exert their antitumoral activities have been defined, including the induction of DNA damage, control of gene expression, and regulation of metabolic pathways, among others. Here, we provided a perspective on the structure and function of G4 ligands with particular emphasis on their potential role as antitumoral agents. In particular, we critically examined the problems associated with the clinical translation of these molecules, trying to highlight the main aspects that should be taken into account during the phases of drug design and development. Indeed, taking advantage of the successes and failures, and the more recent technological progresses in the field, it would be possible to hypothesize the development of these molecules in the future that would represent a valid option for those cancers still missing effective therapies.
RESUMEN
BACKGROUND: Breast Cancer (BC) can be classified, due to its heterogeneity, into multiple subtypes that differ for prognosis and clinical management. Notably, triple negative breast cancer (TNBC) - the most aggressive BC form - is refractory to endocrine and most of the target therapies. In this view, taxane-based therapy still represents the elective strategy for the treatment of this tumor. However, due variability in patients' response, management of TNBC still represents an unmet medical need. Telomeric Binding Factor 2 (TRF2), a key regulator of telomere integrity that is over-expressed in several tumors, including TNBC, has been recently found to plays a role in regulating autophagy, a degradative process that is involved in drug detoxification. Based on these considerations, we pointed, here, at investigating if TRF2, regulating autophagy, can affect tumor sensitivity to therapy. METHODS: Human TNBC cell lines, over-expressing or not TRF2, were subjected to treatment with different taxanes and drug efficacy was tested in terms of autophagic response and cell proliferation. Autophagy was evaluated first biochemically, by measuring the levels of LC3, and then by immunofluorescence analysis of LC3-puncta positive cells. Concerning the proliferation, cells were subjected to colony formation assays associated with western blot and FACS analyses. The obtained results were then confirmed also in mouse models. Finally, the clinical relevance of our findings was established by retrospective analysis on a cohort of TNBC patients subjected to taxane-based neoadjuvant chemotherapy. RESULTS: This study demonstrated that TRF2, inhibiting autophagy, is able to increase the sensitivity of TNBC cells to taxanes. The data, first obtained in in vitro models, were then recapitulated in preclinical mouse models and in a cohort of TNBC patients, definitively demonstrating that TRF2 over-expression enhances the efficacy of taxane-based neoadjuvant therapy in reducing tumor growth and its recurrence upon surgical intervention. CONCLUSIONS: Based on our finding it is possible to conclude that TRF2, already known for its role in promoting tumor formation and progression, might represents an Achilles' heel for cancer. In this view, TRF2 might be exploited as a putative biomarker to predict the response of TNBC patients to taxane-based neoadjuvant chemotherapy.
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Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Estudios Retrospectivos , Taxoides/farmacología , Taxoides/uso terapéutico , Hidrocarburos Aromáticos con Puentes/farmacología , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Línea Celular TumoralRESUMEN
TERF2/TRF2 is a pleiotropic telomeric protein that plays a crucial role in tumor formation and progression through several telomere-dependent and -independent mechanisms. Here, we uncovered a novel function for this protein in regulating the macroautophagic/autophagic process upon different stimuli. By using both biochemical and cell biology approaches, we found that TERF2 binds to the non-histone chromatin-associated protein HMGB1, and this interaction is functional to the nuclear/cytoplasmic protein localization. Specifically, silencing of TERF2 alters the redox status of the cells, further exacerbated upon EBSS nutrient starvation, promoting the cytosolic translocation and the autophagic activity of HMGB1. Conversely, overexpression of wild-type TERF2, but not the mutant unable to bind HMGB1, negatively affects the cytosolic translocation of HMGB1, counteracting the stimulatory effect of EBSS starvation. Moreover, genetic depletion of HMGB1 or treatment with inflachromene, a specific inhibitor of its cytosolic translocation, completely abolished the pro-autophagic activity of TERF2 silencing. In conclusion, our data highlighted a novel mechanism through which TERF2 modulates the autophagic process, thus demonstrating the key role of the telomeric protein in regulating a process that is fundamental, under both physiological and pathological conditions, in defining the fate of the cells.Abbreviations: ALs: autolysosomes; ALT: alternative lengthening of telomeres; ATG: autophagy related; ATM: ATM serine/threonine kinase; CQ: Chloroquine; DCFDA: 2',7'-dichlorofluorescein diacetate; DDR: DNA damage response; DHE: dihydroethidium; EBSS: Earle's balanced salt solution; FACS: fluorescence-activated cell sorting; GFP: green fluorescent protein; EGFP: enhanced green fluorescent protein; GSH: reduced glutathione; GSSG: oxidized glutathione; HMGB1: high mobility group box 1; ICM: inflachromene; IF: immunofluorescence; IP: immunoprecipitation; NAC: N-acetyl-L-cysteine; NHEJ: non-homologous end joining; PLA: proximity ligation assay; RFP: red fluorescent protein; ROS: reactive oxygen species; TIF: telomere-induced foci; TERF2/TRF2: telomeric repeat binding factor 2.
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Proteína HMGB1 , Proteína HMGB1/genética , Daño del ADN , Autofagia/genética , Telómero/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
A focused library of analogs of a lead-like G-quadruplex (G4) targeting compound (4), sharing a furobenzoxazine naphthoquinone core and differing for the pendant groups on the N-atom of the oxazine ring, has been here analyzed with the aim of developing more potent and selective ligands. These molecules have been tested vs. topologically different G4s by the G4-CPG assay, an affinity chromatography-based method for screening putative G4 ligands. The obtained results showed that all these compounds were able to bind several G4 structures, both telomeric and extra-telomeric, thus behaving as multi-target ligands, and two of them fully discriminated G4 vs. duplex DNA. Biological assays proved that almost all the compounds produced effective DNA damage, showing marked antiproliferative effects on tumor cells in the low µM range. Combined analysis of the G4-CPG binding assays and biological data led us to focus on compound S4-5, proved to be less cytotoxic than the parent compound 4 on normal cells. An in-depth biophysical characterization of the binding of S4-5 to different G4s showed that the here identified ligand has higher affinity for the G4s and higher ability to discriminate G4 vs. duplex DNA than 4. Molecular docking studies, in agreement with the NMR data, suggest that S4-5 interacts with the accessible grooves of the target G4 structures, giving clues for its increased G4 vs. duplex selectivity.
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Diseño de Fármacos , G-Cuádruplex/efectos de los fármacos , Sitios de Unión , Proliferación Celular/efectos de los fármacos , Cromatografía de Afinidad , Daño del ADN , Humanos , Ligandos , Naftoquinonas/química , Naftoquinonas/farmacología , Oxazinas/química , Oxazinas/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
Sirtuin 6 (SIRT6) is a member of the NAD+-dependent class III deacetylase sirtuin family, which plays a key role in cancer by controlling transcription, genome stability, telomere integrity, DNA repair, and autophagy. Here we analyzed the molecular and biological effects of UBCS039, the first synthetic SIRT6 activator. Our data demonstrated that UBCS039 induced a time-dependent activation of autophagy in several human tumor cell lines, as evaluated by increased content of the lipidated form of LC3B by western blot and of autophagosomal puncta by microscopy analysis of GFP-LC3. UBCS039-mediated activation of autophagy was strictly dependent on SIRT6 deacetylating activity since the catalytic mutant H133Y failed to activate autophagy. At the molecular level, SIRT6-mediated autophagy was triggered by an increase of ROS levels, which, in turn, resulted in the activation of the AMPK-ULK1-mTOR signaling pathway. Interestingly, antioxidants were able to completely counteract UBCS039-induced autophagy, suggesting that ROS burst had a key role in upstream events leading to autophagy commitment. Finally, sustained activation of SIRT6 resulted in autophagy-related cell death, a process that was markedly attenuated using either a pan caspases inhibitor (zVAD-fmk) or an autophagy inhibitor (CQ). Overall, our results identified UBCS039 as an efficient SIRT6 activator, thereby providing a proof of principle that modulation of the enzyme can influence therapeutic strategy by enhancing autophagy-dependent cell death.
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Autofagia/efectos de los fármacos , Activación Enzimática , Neoplasias/metabolismo , Neoplasias/patología , Pirroles/farmacología , Quinoxalinas/farmacología , Sirtuinas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Autofagosomas/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Ciclo Celular , Proliferación Celular , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pirroles/síntesis química , Quinoxalinas/síntesis química , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant multisystemic disorders caused by expansion of microsatellite repeats. In both forms, the mutant transcripts accumulate in nuclear foci altering the function of alternative splicing regulators which are necessary for the physiological mRNA processing. Missplicing of insulin receptor (IR) gene (INSR) has been associated with insulin resistance, however, it cannot be excluded that post-receptor signalling abnormalities could also contribute to this feature in DM. We have analysed the insulin pathway in skeletal muscle biopsies and in myotube cultures from DM patients to assess whether downstream metabolism might be dysregulated and to better characterize the mechanism inducing insulin resistance. DM skeletal muscle exhibits alterations of basal phosphorylation levels of Akt/PKB, p70S6K, GSK3ß and ERK1/2, suggesting that these changes might be accompanied by a lack of further insulin stimulation. Alterations of insulin pathway have been confirmed on control and DM myotubes expressing fetal INSR isoform (INSR-A). The results indicate that insulin action appears to be lower in DM than in control myotubes in terms of protein activation and glucose uptake. Our data indicate that post-receptor signalling abnormalities might contribute to DM insulin resistance regardless the alteration of INSR splicing.
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Empalme Alternativo , Antígenos CD , Regulación de la Expresión Génica , Resistencia a la Insulina/genética , Distrofia Miotónica , Receptor de Insulina , Transducción de Señal , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/biosíntesis , Antígenos CD/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Receptor de Insulina/biosíntesis , Receptor de Insulina/genéticaRESUMEN
A novel approach to cancer therapeutics is emerging in the field of G-quadruplex (G4) ligands, small molecules designed to stabilize four-stranded structures that can form at telomeres as well as in other genomic sequences, including oncogene promoter sequences, 5'-UTR regions and introns. In this study, we investigated the binding activity of perylene and coronene derivatives PPL3C, CORON and EMICORON to G4 structures formed within the promoter regions of two important cancer-related genes, c-MYC and BCL-2, and their biochemical effects on gene and protein expression. In order to fully characterize the ability of the selected ligands to bind and stabilize the G4 structures originated by the c-MYC and BCL-2 promoter sequences, we performed electrospray ionization mass spectrometry (ESI-MS), Fluorescence Resonance Energy Transfer (FRET) measurements, Circular Dichroism (CD) spectra and polymerase stop assay. Altogether our results showed that the ligands had a high capacity in binding and stabilizing the G4 structures within the c-MYC and BCL-2 promoter sequences in vitro. Notably, when we evaluated by quantitative real-time PCR and western blotting analysis, the effects of treatment with the different G4 ligands on c-MYC and BCL2 expression in a human melanoma cell line, EMICORON appeared the most effective compound in reducing the mRNA and protein levels of both genes. These results encourage to consider EMICORON as a promising example of multimodal class of an antineoplastic drug, affecting different tumor crucial pathways simultaneously: telomere maintenance (as previously described), cell proliferation and apoptosis via down-regulation of both c-MYC and BCL-2 (this paper).
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Secuencia Rica en GC , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Melanoma , Oncogenes , Perileno , Compuestos Policíclicos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas c-myc , Línea Celular Tumoral , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Perileno/farmacocinética , Perileno/farmacología , Compuestos Policíclicos/farmacocinética , Compuestos Policíclicos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
BACKGROUND: Myotonic dystrophy (DM) is a genetic disorder caused by nucleotide repeats expansion. Sudden death represents the main cause of mortality in DM patients. Here, we investigated the relationship between serum cardiac biomarkers with clinical parameters in DM patients. METHODS: Case-control study included 59 DM patients and 22 healthy controls. An additional group of 62 controls with similar cardiac defects to DM were enrolled. RESULTS: NT-proBNP, hs-cTnT and CK levels were significantly increased in DM patients compared to healthy subjects (p=0.0008, p<0.0001, p<0.0001). Also, hs-cTnT levels were significantly higher in DM compared to control group with cardiac defects (p=0.0003). Positive correlation was found between hs-cTnT and hs-cTnI in both DM patients and controls (p=0.019, p=0.002). Independently from the age, the risk of DM disease was positively related to an increase in hs-cTnT (p=0.03). On the contrary, the risk of DM was not related to hs-cTnI, but was evidenced a role of PR interval (p=0.03) and CK (p=0.08). CONCLUSIONS: The levels of hs-cTnT were significantly higher in DM patients. Analysis, with anti-cTnT, shows that this increase might be linked to heart problems. This last finding suggests that hs-cTnT might represent a helpful serum biomarker to "predict" cardiac risk in DM disease.
Asunto(s)
Cardiopatías/sangre , Cardiopatías/diagnóstico , Distrofia Miotónica/sangre , Distrofia Miotónica/complicaciones , Troponina T/sangre , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Cardiopatías/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
The pentacyclic acridinium salt RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate, compound 1) is one of the most interesting DNA G-quadruplex binding molecules due to its high efficacy in tumor cell growth inhibition both in in vitro models and in vivo against human tumor xenografts in combination with conventional chemotherapeutics. Despite compound 1 having desirable chemical and pharmaceutical properties, its potential as a therapeutic agent is compromised by off-target effects on cardiovascular physiology. In this paper we report a new series of structurally-related compounds which were developed in an attempt to minimize its off-target profile, but maintaining the same favorable chemical and pharmacological features of the lead compound. By performing a comparative analysis it was possible to identify which derivatives had the following properties: (i) to show a reduced capacity in respect to compound 1 to inhibit the hERG tail current tested in a patch clamp assay and/or to interact with the human recombinant ß2 receptor; (ii) to maintain both a good G4-binding affinity and cancer cell selectivity; and (iii) to trigger DNA damage with specific telomere uncapping. These studies allowed us to identify a novel G4-stabilizing molecule, compound 8, being characterized by reduced off-target effects and potent telomere on-target properties compared to the prototypic compound 1. Moreover, compound 8 shares with compound 1 the same molecular mode of action and an anti-tumour activity specifically restricted to replicating cells, as evident with its particularly efficient activity in combination therapy with a topoisomerase I inhibitor. In conclusion, we have identified a new pentacyclic derivative 8 having suitable properties to be the focus of further investigations as a clinical candidate for cancer therapy.
Asunto(s)
Acridinas/farmacología , Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Daño del ADN , Diseño de Fármacos , Telómero/efectos de los fármacos , Acridinas/química , Acridinas/toxicidad , Antineoplásicos/toxicidad , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Relación Dosis-Respuesta a Droga , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Células HT29 , Humanos , Ligandos , Potenciales de la Membrana , Estructura Molecular , Receptores Adrenérgicos beta 2/efectos de los fármacos , Receptores Adrenérgicos beta 2/metabolismo , Relación Estructura-Actividad , Telómero/genética , Telómero/metabolismoRESUMEN
Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment. The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant ß2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay. Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with ß2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities. Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment.
Asunto(s)
Acridinas/química , Acridinas/farmacología , G-Cuádruplex , Telómero/metabolismo , Acridinas/síntesis química , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cobayas , Humanos , Ligandos , Telomerasa/antagonistas & inhibidoresRESUMEN
Targeting of DNA secondary structures, such as G-quadruplexes, is now considered an appealing opportunity for drug intervention in anticancer therapy. So far, efforts made in the discovery of chemotypes able to target G-quadruplexes mainly succeeded in the identification of a number of polyaromatic compounds featuring end-stacking binding properties. Against this general trend, we were persuaded that the G-quadruplex grooves can recognize molecular entities with better drug-like and selectivity properties. From this idea, a set of small molecules was identified and the structural features responsible for G-quadruplex recognition were delineated. These compounds were demonstrated to have enhanced affinity and selectivity for the G-quadruplex over the duplex structure. Their ability to induce selective DNA damage at telomeric level and to induction of apoptosis and senescence on tumor cells is herein experimentally proven.