RESUMEN
Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site.
Asunto(s)
Replicación del ADN , ADN Superhelicoidal , Telómero , Humanos , Telómero/metabolismo , ADN Superhelicoidal/metabolismo , Sitios Frágiles del Cromosoma , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Conformación de Ácido Nucleico , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismoRESUMEN
Topoisomerase (Top) inhibitors used in clinical cancer treatments are limited because of their toxicity and severe side effects. Noteworthily, Top1/2 dual inhibitors overcome the compensatory effect between Top1 and 2 inhibitors to exhibit stronger antitumor efficacies. In this study, a series of indolo[3,2-c]isoquinoline derivatives were designed as Top1/2 dual inhibitors possessing apparent antiproliferative activities. Mechanistic studies indicated that the optimal compounds 23 and 31 with increasing reactive oxygen species levels damage DNA, inducing both cancer cell apoptosis and cycle arrest. Importantly, the results of the toxicity studies showed that compounds 23 and 31 possessed good oral safety profiles. In xenograft models, compound 23 exhibited remarkable antitumor potency, which was superior to the clinical Top inhibitors irinotecan and etoposide. Overall, this work highlights the therapeutic potential and safety profile of compound 23 as a Top1/2 dual inhibitor in tumor therapy and provides valuable lead compounds for further development of Top inhibitors.
Asunto(s)
Antineoplásicos , ADN-Topoisomerasas de Tipo II , Isoquinolinas , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa II , Humanos , Animales , Isoquinolinas/farmacología , Isoquinolinas/química , Isoquinolinas/uso terapéutico , Isoquinolinas/síntesis química , Isoquinolinas/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Antineoplásicos/síntesis química , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/uso terapéutico , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa I/uso terapéutico , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa I/síntesis química , Administración Oral , ADN-Topoisomerasas de Tipo II/metabolismo , Línea Celular Tumoral , Relación Estructura-Actividad , Ratones , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Antitumor por Modelo de Xenoinjerto , Indoles/farmacología , Indoles/química , Indoles/uso terapéutico , Ratones Desnudos , Descubrimiento de Drogas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Since the report of "DNA untwisting" activity in 1972, â¼50 years of research has revealed seven topoisomerases in humans (TOP1, TOP1mt, TOP2α, TOP2ß, TOP3α, TOP3ß and Spo11). These conserved regulators of DNA topology catalyze controlled breakage to the DNA backbone to relieve the torsional stress that accumulates during essential DNA transactions including DNA replication, transcription, and DNA repair. Each topoisomerase-catalyzed reaction involves the formation of a topoisomerase cleavage complex (TOPcc), a covalent protein-DNA reaction intermediate formed between the DNA phosphodiester backbone and a topoisomerase catalytic tyrosine residue. A variety of perturbations to topoisomerase reaction cycles can trigger failure of the enzyme to re-ligate the broken DNA strand(s), thereby generating topoisomerase DNA-protein crosslinks (TOP-DPC). TOP-DPCs pose unique threats to genomic integrity. These complex lesions are comprised of structurally diverse protein components covalently linked to genomic DNA, which are bulky DNA adducts that can directly impact progression of the transcription and DNA replication apparatus. A variety of genome maintenance pathways have evolved to recognize and resolve TOP-DPCs. Eukaryotic cells harbor tyrosyl DNA phosphodiesterases (TDPs) that directly reverse 3'-phosphotyrosyl (TDP1) and 5'-phoshotyrosyl (TDP2) protein-DNA linkages. The broad specificity Mre11-Rad50-Nbs1 and APE2 nucleases are also critical for mitigating topoisomerase-generated DNA damage. These DNA-protein crosslink metabolizing enzymes are further enabled by proteolytic degradation, with the proteasome, Spartan, GCNA, Ddi2, and FAM111A proteases implicated thus far. Strategies to target, unfold, and degrade the protein component of TOP-DPCs have evolved as well. Here we survey mechanisms for addressing Topoisomerase 1 (TOP1) and Topoisomerase 2 (TOP2) DPCs, highlighting systems for which molecular structure information has illuminated function of these critical DNA damage response pathways.
Asunto(s)
Reparación del ADN , Humanos , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo I/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , ADN-Topoisomerasas/metabolismo , Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Replicación del ADNRESUMEN
Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability and a number of clinically important anticancer and antibacterial drugs, e.g. quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50-fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e. negative versus positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.
Asunto(s)
División del ADN , Girasa de ADN , Topoisomerasa de ADN IV , ADN-Topoisomerasas de Tipo II , Escherichia coli , Escherichia coli/genética , Escherichia coli/enzimología , Girasa de ADN/metabolismo , Girasa de ADN/genética , Girasa de ADN/química , Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/química , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Análisis de Secuencia de ADN/métodos , ADN Superhelicoidal/metabolismo , ADN Superhelicoidal/química , Ciprofloxacina/farmacología , Secuenciación de Nucleótidos de Alto Rendimiento , ADN/metabolismo , ADN/químicaRESUMEN
Almost half of all medicines approved by the U.S. Food and Drug Administration have been found to be developed based on inspiration from natural products (NPs). Here, we report a novel strategy of scaffold overlaying of scaffold-hopped analogs of bioactive flavones and isoflavones and installation of drug-privileged motifs, which has led to discovery of anticancer agents that surpass the functional efficiency of the original NPs. The analogs, 2,3-diaryl-pyridopyrimidin-4-imine/ones were efficiently synthesized by an approach of a nitrile-stabilized quaternary ammonium ylide as masked synthon and Pd-catalyzed activation-arylation methods. Compared to the NPs, these NP-analogs exhibited differentiated functions; dual inhibition of human topoisomerase-II (hTopo-II) enzyme and tubulin polymerization, and pronounced antiproliferative effect against various cancer cell lines, including numerous drug-resistant cancer cells. The most active compound 5l displayed significant inhibition of migration ability of cancer cells and blocked G1/S phase transition in cell cycle. Compound 5l caused pronounced effect in expression patterns of various key cell cycle regulatory proteins; up-regulation of apoptotic proteins, Bax, Caspase 3 and p53, and down-regulation of apoptosis-inhibiting proteins, BcL-xL, Cyclin D1, Cyclin E1 and NF-κB, which indicates high efficiency of the molecule 5l in apoptosis-signal axis interfering potential. Cheminformatics analysis revealed that 2,3-diaryl-pyridopyrimidin-4-imine/ones occupy a distinctive drug-relevant chemical space that is seldom represented by natural products and good physicochemical, ADMET and pharmacokinetic-relevant profile. Together, the anticancer potential of the investigated analogs was found to be much more efficient compared to the original natural products and two anticancer drugs, Etoposide (hTopo-II inhibitor) and 5-Flurouracile (5-FU).
Asunto(s)
Antineoplásicos , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Flavonoides/química , Flavonoides/farmacología , Flavonoides/síntesis química , Iminas/química , Iminas/farmacología , Iminas/síntesis química , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacología , Moduladores de Tubulina/química , Moduladores de Tubulina/síntesis química , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/farmacologíaRESUMEN
OBJECTIVES: The present study used single-cell RNA sequencing (scRNA-seq) to characterize the cellular composition of ovarian carcinosarcoma (OCS) and identify its molecular characteristics. METHODS: scRNA-seq was performed in resected primary OCS for an in-depth analysis of tumor cells and the tumor microenvironment. Immunohistochemistry staining was used for validation. The scRNA-seq data of OCS were compared with those of high-grade serous ovarian carcinoma (HGSOC) tumors and other OCS tumors. RESULTS: Both malignant epithelial and malignant mesenchymal cells were observed in the OCS patient of this study. We identified four epithelial cell subclusters with different biological roles. Among them, epithelial subcluster 4 presented high levels of breast cancer type 1 susceptibility protein homolog (BRCA1) and DNA topoisomerase 2-α (TOP2A) expression and was related to drug resistance and cell cycle. We analyzed the interaction between epithelial and mesenchymal cells and found that fibroblast growth factor (FGF) and pleiotrophin (PTN) signalings were the main pathways contributing to communication between these cells. Moreover, we compared the malignant epithelial and mesenchymal cells of this OCS tumor with our previous published HGSOC scRNA-seq data and OCS data. All the epithelial subclusters in the OCS tumor could be found in the HGSOC samples. Notably, the mesenchymal subcluster C14 exhibited specific expression patterns in the OCS tumor, characterized by elevated expression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1), collagen type XXIII α1 chain (COL23A1), cholecystokinin (CCK), bone morphogenetic protein 7 (BMP7), PTN, Wnt inhibitory factor 1 (WIF1), and insulin-like growth factor 2 (IGF2). Moreover, this subcluster showed distinct characteristics when compared with both another previously published OCS tumor and normal ovarian tissue. CONCLUSIONS: This study provides the single-cell transcriptomics signature of human OCS, which constitutes a new resource for elucidating OCS diversity.
Asunto(s)
Carcinosarcoma , ADN-Topoisomerasas de Tipo II , Neoplasias Ováricas , Análisis de la Célula Individual , Transcriptoma , Humanos , Femenino , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Carcinosarcoma/genética , Carcinosarcoma/metabolismo , Carcinosarcoma/patología , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Microambiente Tumoral , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Citocinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Análisis de Secuencia de ARN , Persona de Mediana Edad , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Cistadenocarcinoma Seroso/metabolismo , Proteínas de Unión a Poli-ADP-RibosaRESUMEN
The ability to catalyze reversible DNA cleavage and religation is central to topoisomerases' role in regulating DNA topology. In type IIA topoisomerases (Top2), the formation of its DNA cleavage-religation center is driven by DNA-binding-induced structural rearrangements. These changes optimally position key catalytic modules, such as the active site tyrosine of the WHD domain and metal ion(s) chelated by the TOPRIM domain, around the scissile phosphodiester bond to perform reversible transesterification. To understand this assembly process in detail, we report the catalytic core structures of human Top2α and Top2ß in an on-pathway conformational state. This state features an in trans formation of an interface between the Tower and opposing TOPRIM domain, revealing a groove for accommodating incoming G-segment DNA. Structural superimposition further unveils how subsequent DNA-binding-induced disengagement of the TOPRIM and Tower domains allows a firm grasp of the bound DNA for cleavage/religation. Notably, we identified a previously undocumented protein-DNA interaction, formed between an arginine-capped C-terminus of an α-helix in the TOPRIM domain and the DNA backbone, significantly contributing to Top2 function. This work uncovers a previously unrecognized role of the Tower domain, highlighting its involvement in anchoring and releasing the TOPRIM domain, thus priming Top2 for DNA binding and cleavage.
Asunto(s)
División del ADN , ADN-Topoisomerasas de Tipo II , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/química , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ADN/química , ADN/metabolismo , Dominio Catalítico , Modelos Moleculares , Unión Proteica , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Cristalografía por Rayos XRESUMEN
Targeting inter-duplex junctions in catenated DNA with bidirectional bis-intercalators is a potential strategy for enhancing anticancer effects. In this study, we used d(CGTATACG)2, which forms a tetraplex base-pair junction that resembles the DNA-DNA contact structure, as a model target for two alkyl-linked diaminoacridine bis-intercalators, DA4 and DA5. Cross-linking of the junction site by the bis-intercalators induced substantial structural changes in the DNA, transforming it from a B-form helical end-to-end junction to an over-wounded side-by-side inter-duplex conformation with A-DNA characteristics and curvature. These structural perturbations facilitated the angled intercalation of DA4 and DA5 with propeller geometry into two adjacent duplexes. The addition of a single carbon to the DA5 linker caused a bend that aligned its chromophores with CpG sites, enabling continuous stacking and specific water-mediated interactions at the inter-duplex contacts. Furthermore, we have shown that the different topological changes induced by DA4 and DA5 lead to the inhibition of topoisomerase 2 activities, which may account for their antitumor effects. Thus, this study lays the foundations for bis-intercalators targeting biologically relevant DNA-DNA contact structures for anticancer drug development.
Asunto(s)
Antineoplásicos , ADN , Sustancias Intercalantes , Conformación de Ácido Nucleico , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Humanos , ADN/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/química , Línea Celular Tumoral , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Aim: To synthesize new hybrid cinnamic acids (10a, 10b and 11) and ester derivatives (7, 8 and 9) and investigate their anti-breast cancer activities.Materials & methods: Compounds 7-11 were evaluated (in vitro) for their cytotoxic activities against the MCF-7 cell line. A flow cytometry examination was performed. Protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), topoisomerase II and caspase-9 were measured by qRT-PCR. Molecular docking studies were conducted.Results: Several components were discovered to be active, mainly component 11, which induced arrest in the cell cycle at phase S, greatly decreased the expression of Nrf2 and topoisomerase II; and upregulated the expression of caspase-9.Conclusion: The newly thiohydantoin-cinnamic acid hybrids can contribute to creating promising candidates for cancer drugs.
[Box: see text].
Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Cinamatos , Simulación del Acoplamiento Molecular , Humanos , Cinamatos/química , Cinamatos/farmacología , Cinamatos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Femenino , Células MCF-7 , Tiohidantoínas/farmacología , Tiohidantoínas/química , Tiohidantoínas/síntesis química , Ensayos de Selección de Medicamentos Antitumorales , Proliferación Celular/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Relación Estructura-Actividad , Estructura Molecular , ADN-Topoisomerasas de Tipo II/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 9/metabolismoRESUMEN
DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.
Asunto(s)
ADN-Topoisomerasas de Tipo II , Heterocromatina , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Heterocromatina/metabolismo , Animales , Inhibidores de Topoisomerasa II/farmacología , Secuencias Repetitivas de Ácidos Nucleicos/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Replicación del ADN , ADN Superhelicoidal/metabolismo , ADN Superhelicoidal/química , Humanos , Ratones , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN/metabolismo , ADN/química , InterfaseRESUMEN
The discovery of novel oncotargets for glioma is of immense significance. We here explored the expression patterns, biological functions, and underlying mechanisms associated with ORC6 (origin recognition complex 6) in glioma. Through the bioinformatics analyses, we found a significant increase in ORC6 expression within human glioma tissues, correlating with poorer overall survival, higher tumor grade, and wild-type isocitrate dehydrogenase status. Additionally, ORC6 overexpression is detected in glioma tissues obtained from locally-treated patients and across various primary/established glioma cells. Further bioinformatics scrutiny revealed that genes co-expressed with ORC6 are enriched in multiple signaling cascades linked to cancer. In primary and immortalized (A172) glioma cells, depleting ORC6 using specific shRNA or Cas9-sgRNA knockout (KO) significantly decreased cell viability and proliferation, disrupted cell cycle progression and mobility, and triggered apoptosis. Conversely, enhancing ORC6 expression via a lentiviral construct augmented malignant behaviors in human glioma cells. ORC6 emerged as a crucial regulator for the expression of key oncogenic genes, including Cyclin A2, Cyclin B2, and DNA topoisomerase II (TOP2A), within glioma cells. Silencing or KO of ORC6 reduced the mRNA and protein levels of these genes, while overexpression of ORC6 increased their expression in primary glioma cells. Bioinformatics analyses further identified RBPJ as a potential transcription factor of ORC6. RBPJ shRNA decreased ORC6 expression in primary glioma cells, while its overexpression increased it. Additionally, significantly enhanced binding between the RBPJ protein and the proposed ORC6 promoter region was detected in glioma tissues and cells. In vivo experiments demonstrated a significant reduction in the growth of patient-derived glioma xenografts in the mouse brain subsequent to ORC6 KO. ORC6 depletion, inhibited proliferation, decreased expression of Cyclin A2/B2/TOP2A, and increased apoptosis were detected within these ORC6 KO intracranial glioma xenografts. Altogether, RBPJ-driven ORC6 overexpression promotes glioma cell growth, underscoring its significance as a promising therapeutic target.
Asunto(s)
Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioma , Complejo de Reconocimiento del Origen , Animales , Humanos , Masculino , Ratones , Apoptosis/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Ciclina A2/metabolismo , Ciclina A2/genética , Ciclina B2/metabolismo , Ciclina B2/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Glioma/genética , Glioma/patología , Glioma/metabolismo , Ratones Desnudos , Complejo de Reconocimiento del Origen/metabolismo , Complejo de Reconocimiento del Origen/genéticaRESUMEN
Trichomes are specialized epidermal structures that protect plants from biotic and abiotic stresses by synthesizing, storing, and secreting defensive compounds. This study investigates the role of the Gossypium arboreum DNA topoisomerase VI subunit B gene (GaTOP6B) in trichome development and branching. Sequence alignment revealed a high similarity between GaTOP6B and AtTOP6B, suggesting a conserved function in trichome regulation. Although AtTOP6B acts as a positive regulator of trichome development, functional analyses showed contrasting effects: Virus-induced gene silencing (VIGS) of GaTOP6B in cotton increased trichome density, while its overexpression in Arabidopsis decreased trichome density but enhanced branching. This demonstrates that GaTOP6B negatively regulates trichome number, indicating species-specific roles in trichome initiation and branching between cotton and Arabidopsis. Overexpression of the GaTOP6B promotes jasmonic acid synthesis, which in turn inhibits the G1/S or G2/M transitions, stalling the cell cycle. On the other hand, it suppresses brassinolide synthesis and signaling while promoting cytokinin degradation, further inhibiting mitosis. These hormonal interactions facilitate the transition of cells from the mitotic cycle to the endoreduplication cycle. As the level of endoreduplication increases, trichomes develop an increased number of branches. These findings highlight GaTOP6B's critical role as a regulator of trichome development, providing new genetic targets for improving cotton varieties in terms of enhanced adaptability and resilience.
Asunto(s)
Arabidopsis , Ciclopentanos , Endorreduplicación , Regulación de la Expresión Génica de las Plantas , Gossypium , Oxilipinas , Proteínas de Plantas , Tricomas , Tricomas/genética , Tricomas/crecimiento & desarrollo , Tricomas/metabolismo , Gossypium/genética , Gossypium/crecimiento & desarrollo , Gossypium/metabolismo , Ciclopentanos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Oxilipinas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Endorreduplicación/genética , Brasinoesteroides/metabolismo , Plantas Modificadas Genéticamente , Genes de Plantas , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Esteroides HeterocíclicosRESUMEN
We have conducted an experimental and computational evaluation of new doxorubicin (4a-c) and ß-lapachone (5a-c) analogs. These novel anticancer analogs were previously synthesized, but had not been tested or characterized until now. We have evaluated their antiproliferative and DNA cleavage inhibition properties using breast (MCF-7 and MDA-MB-231) and prostate (PC3) cancer cell lines. Additionally, cell cycle analysis was performed using flow cytometry. Computational studies, including molecular docking, pharmacokinetic properties, and an analysis of DFT and QTAIM chemical descriptors, were performed to gain insights into the electronic structure and elucidate the molecular binding of the new ß-lapachone and doxorubicin analogs with a DNA sequence and Topoisomerase II (Topo II)α. Our results show that 4a analog displays the highest antiproliferative activity in cancer cell lines by inducing cell death. We observed that stacking interactions and hydrogen bonding are essential to stabilize the molecule-DNA-Topo IIα complex. Moreover, 4a and 5a analogs inhibited Topo's DNA cleavage activity. Pharmacodynamic results indicated that studied molecules have favorable adsorption and permeability properties. The calculated chemical descriptors indicate that electron accumulation in quinone rings is relevant to the reactivity and biological activity. Based on our results, 4a is a strong candidate for becoming an anticancer drug.
Asunto(s)
Antineoplásicos , Proliferación Celular , ADN-Topoisomerasas de Tipo II , Doxorrubicina , Simulación del Acoplamiento Molecular , Naftoquinonas , Naftoquinonas/química , Naftoquinonas/farmacología , Humanos , Doxorrubicina/farmacología , Doxorrubicina/química , ADN-Topoisomerasas de Tipo II/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células MCF-7 , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/metabolismo , División del ADN/efectos de los fármacosRESUMEN
Pausing of RNA polymerase II (Pol II) at transcription start sites (TSSs) primes target genes for productive elongation. Coincidentally, DNA double-strand breaks (DSBs) enrich at highly transcribed and Pol II-paused genes, although their interplay remains undefined. Using androgen receptor (AR) signaling as a model, we have uncovered AR-interacting protein 4 (ARIP4) helicase as a driver of androgen-dependent transcription induction. Chromatin immunoprecipitation sequencing analysis revealed that ARIP4 preferentially co-occupies TSSs with paused Pol II. Moreover, we found that ARIP4 complexes with topoisomerase II beta and mediates transient DSB formation upon hormone stimulation. Accordingly, ARIP4 deficiency compromised release of paused Pol II and resulted in R-loop accumulation at a panel of highly transcribed AR target genes. Last, we showed that ARIP4 binds and unwinds R-loops in vitro and that its expression positively correlates with prostate cancer progression. We propose that androgen stimulation triggers ARIP4-mediated unwinding of R-loops at TSSs, enforcing Pol II pause release to effectively drive an androgen-dependent expression program.
Asunto(s)
Andrógenos , Neoplasias de la Próstata , Estructuras R-Loop , ARN Polimerasa II , Receptores Androgénicos , Humanos , Andrógenos/metabolismo , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Transcripción Genética , Roturas del ADN de Doble Cadena , Sitio de Iniciación de la Transcripción , Regulación Neoplásica de la Expresión Génica , Unión Proteica , Activación TranscripcionalRESUMEN
A series of tetrahydrobenzo[b]thiophene derivatives was designed and synthesized as dual topoisomerase (Topo) I/II inhibitors implicating potential DNA intercalation. Ethyl-2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylate (1) was prepared by modification of the Gewald reaction procedure using a Fe2O3 nanocatalyst and then it was used as a building block for the synthesis of tetrahydrobenzo[b]thiophene candidates (2-14). Interestingly, compound 14 showed the best cytotoxic potential against hepatocellular, colorectal, and breast cancer cell lines (IC50 = 7.79, 8.10, and 3.53 µM), respectively, surpassing doxorubicin at breast cancer (IC50 = 4.17 µM). Meanwhile, the Topo I and II inhibition assay displayed that compound 3 could exhibit the best inhibitory potential among the investigated candidates (IC50 = 25.26 and 10.01 nM), respectively, in comparison to camptothecin (IC50 = 28.34 nM) and doxorubicin (IC50 = 11.01 nM), as reference standards. In addition, the DNA intercalation assay showed that compound 14 could display the best binding affinity with an IC50 value of 77.82 µM in comparison to doxorubicin (IC50 = 58.03 µM). Furthermore, cell cycle and apoptosis analyses described that compound 3 prompts the G1 phase arrest in michigan cancer foundation-7 cancer cells and increases the apoptosis ratio by 29.31% with respect to untreated cells (2.25%). Additionally, the conducted molecular docking assured the promising binding of the investigated members toward Topo I and II with potential DNA intercalation. Accordingly, the synthesized compounds could be treated as promising anticancer candidates for future optimization.
Asunto(s)
Antineoplásicos , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sustancias Intercalantes , Tiofenos , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa II , Humanos , Tiofenos/farmacología , Tiofenos/síntesis química , Tiofenos/química , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa I/síntesis química , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/química , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Relación Estructura-Actividad , Sustancias Intercalantes/farmacología , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/química , Estructura Molecular , Simulación del Acoplamiento Molecular , Relación Dosis-Respuesta a Droga , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Apoptosis/efectos de los fármacos , ADN , ADN-Topoisomerasas de Tipo I/metabolismo , FarmacóforoRESUMEN
DNA Topoisomerase IIα (Top2A) is a nuclear enzyme that is a cancer drug target, and there is interest in identifying novel sites on the enzyme to inhibit cancer cells more selectively and to reduce off-target toxicity. The C-terminal domain (CTD) is one potential target, but it is an intrinsically disordered domain, which prevents structural analysis. Therefore, we set out to analyze the sequence of Top2A from 105 species using bioinformatic analysis, including the PSICalc algorithm, Shannon entropy analysis, and other approaches. Our results demonstrate that large (10th-order) interdependent clusters are found including non-proximal positions across the major domains of Top2A. Further, CTD-specific clusters of the third, fourth, and fifth order, including positions that had been previously analyzed via mutation and biochemical assays, were identified. Some of these clusters coincided with positions that, when mutated, either increased or decreased relaxation activity. Finally, sites of low Shannon entropy (i.e., low variation in amino acids at a given site) were identified and mapped as key positions in the CTD. Included in the low-entropy sites are phosphorylation sites and charged positions. Together, these results help to build a clearer picture of the critical positions in the CTD and provide potential sites/regions for further analysis.
Asunto(s)
Biología Computacional , ADN-Topoisomerasas de Tipo II , Dominios Proteicos , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/química , Biología Computacional/métodos , Humanos , Entropía , Secuencia de Aminoácidos , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/química , FosforilaciónRESUMEN
BACKGROUND/AIM: Hyperthermia represents an adjuvant local anticancer strategy which relies on the increase of temperature beyond the physiological level. In this study, we investigated the anticancer potential of Fe3O4 and Fe3O4core Aushell nanoparticles as hyperthermic agents in terms of cytotoxicity and studied the expression of cellular markers of proliferation (changes in mRNA levels via real-time polymerase chain reaction). MATERIALS AND METHODS: The human breast cancer cell line SK-BR-1 was incubated with either Fe3O4 or Fe3O4core Aushell nanoparticles stabilized with tryptophan, prior to hyperthermia treatment. The normal HEK293 cell line was used as a control. Toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to estimate possible toxic effects of the tested nanoparticles. After RNA extraction and cDNA synthesis, mRNA expression of three indicators of proliferation, namely marker of proliferation Ki-67, DNA topoisomerase II alpha (TOP2A) and TPX2 microtubule nucleation factor (TPX2), was investigated. RESULTS: At each concentration tested, Fe3O4core Aushell nanoparticles showed greater toxicity compared to Fe3O4, while SK-BR-3 cells were more susceptible to their cytotoxic effects compared to the HEK293 cell line. The expression of Ki-67, TOP2A and TPX2 was reduced in SK-BR-3 cells by both Fe3O4 or Fe3O4core Aushell nanoparticles compared to untreated cells, while the only observed change in HEK293 cells was the up-regulation of TOP2A. CONCLUSION: Both Fe3O4core Aushell and Fe3O4 NPs exhibit increased cytotoxicity to the cancer cell line tested (SK-BR-3) compared to HEK293 cells. The down-regulation in SK-BR-3 cells of the three proliferative markers studied, Ki-67, TOP2A and TPX2, after incubation with NPs suggests that cells that survived thermal destruction were not actively proliferating.
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Neoplasias de la Mama , Proteínas de Ciclo Celular , Proliferación Celular , ADN-Topoisomerasas de Tipo II , Hipertermia Inducida , Antígeno Ki-67 , Proteínas Asociadas a Microtúbulos , Proteínas de Unión a Poli-ADP-Ribosa , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Proliferación Celular/efectos de los fármacos , Hipertermia Inducida/métodos , Antígeno Ki-67/metabolismo , Antígeno Ki-67/genética , Línea Celular Tumoral , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Femenino , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Células HEK293 , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genéticaRESUMEN
Meiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double-strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well-defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of Caenorhabditis elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5'-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the WT TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Segregación Cromosómica , ADN-Topoisomerasas de Tipo II , Espermatogénesis , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Espermatogénesis/genética , Masculino , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Meiosis , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , MutaciónRESUMEN
BACKGROUND/AIM: Pulsed electromagnetic field (PEMF) stimulation enhances the efficacy of several anticancer drugs. Doxorubicin is an anticancer drug used to treat various types of cancer, including breast cancer. However, the effect of PEMF stimulation on the efficacy of doxorubicin and the underlying mechanisms remain unclear. Thus, this study aimed to investigate the effect of PEMF stimulation on the anticancer activity of doxorubicin in MDA-MB-231 human breast cancer cells. MATERIALS AND METHODS: MDA-MB-231 cells were seeded and allowed to incubate for 48 h. The cells were treated with doxorubicin, cisplatin, 5-fluorouracil, or paclitaxel for 48 h. Subsequently, the cells were stimulated with a 60-min PEMF session thrice a day (with an interval of 4 h between each session) for 24 or 48 h. Cell viability was assessed by trypan blue dye exclusion assay and cell-cycle analysis was analyzed by flow cytometry. Molecular mechanisms involved in late G2 arrest were confirmed by a western blot assay and confocal microscopy. RESULTS: MDA-MB-231 cells treated with a combination of doxorubicin and PEMF had remarkably lower viability than those treated with doxorubicin alone. PEMF stimulation increased doxorubicin-induced cell-cycle arrest in the late G2 phase by suppressing cyclin-dependent kinase 1 (CDK1) activity through the enhancement of myelin transcription factor 1 (MYT1) expression, cell division cycle 25C (CDC25C) phosphorylation, and stratifin (14-3-3σ) expression. PEMF also increased doxorubicin-induced DNA damage by inhibiting DNA topoisomerase II alpha (TOP2A). CONCLUSION: These findings support the use of PEMF stimulation as an adjuvant to strengthen the antiproliferative effect of doxorubicin on breast cancer cells.
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Neoplasias de la Mama , Doxorrubicina , Humanos , Doxorrubicina/farmacología , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Femenino , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Campos Electromagnéticos , ADN-Topoisomerasas de Tipo II/metabolismo , Proliferación Celular/efectos de los fármacos , Paclitaxel/farmacología , Fluorouracilo/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Fosfatasas cdc25/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismoRESUMEN
Meningioma is the most common primary brain tumor and many studies have evaluated numerous biomarkers for their prognostic value, often with inconsistent results. Currently, no reliable biomarkers are available to predict the survival, recurrence, and progression of meningioma patients in clinical practice. This study aims to evaluate the prognostic value of immunohistochemistry-based (IHC) biomarkers of meningioma patients. A systematic literature search was conducted up to November 2023 on PubMed, CENTRAL, CINAHL Plus, and Scopus databases. Two authors independently reviewed the identified relevant studies, extracted data, and assessed the risk of bias of the studies included. Meta-analyses were performed with the hazard ratio (HR) and 95% confidence interval (CI) of overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS). The risk of bias in the included studies was evaluated using the Quality in Prognosis Studies (QUIPS) tool. A total of 100 studies with 16,745 patients were included in this review. As the promising markers to predict OS of meningioma patients, Ki-67/MIB-1 (HR = 1.03, 95%CI 1.02 to 1.05) was identified to associate with poor prognosis of the patients. Overexpression of cyclin A (HR = 4.91, 95%CI 1.38 to 17.44), topoisomerase II α (TOP2A) (HR = 4.90, 95%CI 2.96 to 8.12), p53 (HR = 2.40, 95%CI 1.73 to 3.34), vascular endothelial growth factor (VEGF) (HR = 1.61, 95%CI 1.36 to 1.90), and Ki-67 (HR = 1.33, 95%CI 1.21 to 1.46), were identified also as unfavorable prognostic biomarkers for poor RFS of meningioma patients. Conversely, positive progesterone receptor (PR) and p21 staining were associated with longer RFS and are considered biomarkers of favorable prognosis of meningioma patients (HR = 0.60, 95% CI 0.41 to 0.88 and HR = 1.89, 95%CI 1.11 to 3.20). Additionally, high expression of Ki-67 was identified as a prognosis biomarker for poor PFS of meningioma patients (HR = 1.02, 95%CI 1.00 to 1.04). Although only in single studies, KPNA2, CDK6, Cox-2, MCM7 and PCNA are proposed as additional markers with high expression that are related with poor prognosis of meningioma patients. In conclusion, the results of the meta-analysis demonstrated that PR, cyclin A, TOP2A, p21, p53, VEGF and Ki-67 are either positively or negatively associated with survival of meningioma patients and might be useful biomarkers to assess the prognosis.