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1.
Nat Commun ; 12(1): 2962, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016969

RESUMO

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.


Assuntos
DNA Topoisomerases Tipo II/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , Regulação Alostérica , Animais , Domínio Catalítico , Linhagem Celular , Microscopia Crioeletrônica , DNA/metabolismo , DNA/ultraestrutura , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/isolamento & purificação , DNA Topoisomerases Tipo II/metabolismo , Humanos , Mesocricetus , Modelos Moleculares , Nucleoproteínas , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/isolamento & purificação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
2.
Nat Commun ; 12(1): 2917, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006877

RESUMO

Topoisomerase II (topo II) is one of the six proteins essential for mitotic chromatid reconstitution in vitro. It is not fully understood, however, mechanistically how this enzyme regulates this process. In an attempt to further refine the reconstitution assay, we have found that chromosomal binding of Xenopus laevis topo IIα is sensitive to buffer conditions and depends on its C-terminal domain (CTD). Enzymological assays using circular DNA substrates supports the idea that topo IIα first resolves inter-chromatid entanglements to drive individualization and then generates intra-chromatid entanglements to promote thickening. Importantly, only the latter process requires the CTD. By using frog egg extracts, we also show that the CTD contributes to proper formation of nucleosome-depleted chromatids by competing with a linker histone for non-nucleosomal DNA. Our results demonstrate that topo IIα utilizes its CTD to deliver the enzymatic core to crowded environments created during mitotic chromatid assembly, thereby fine-tuning this process.


Assuntos
Cromátides/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Sítios de Ligação/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromátides/genética , Segregação de Cromossomos/genética , DNA/genética , DNA/metabolismo , DNA Topoisomerases Tipo II/genética , Feminino , Histonas/metabolismo , Masculino , Mitose/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Espermatozoides/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética
3.
Molecules ; 26(6)2021 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-33801057

RESUMO

A cannabinoid anticancer para-quinone, HU-331, which was synthesized by our group five decades ago, was shown to have very high efficacy against human cancer cell lines in-vitro and against in-vivo grafts of human tumors in nude mice. The main mechanism was topoisomerase IIα catalytic inhibition. Later, several groups synthesized related compounds. In the present presentation, we review the publications on compounds synthesized on the basis of HU-331, summarize their published activities and mechanisms of action and report the synthesis and action of novel quinones, thus expanding the structure-activity relationship in these series.


Assuntos
Canabidiol/análogos & derivados , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Quinonas , Inibidores da Topoisomerase II , Animais , Canabidiol/química , Canabidiol/uso terapêutico , DNA Topoisomerases Tipo II/metabolismo , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/enzimologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Quinonas/química , Quinonas/uso terapêutico , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/uso terapêutico
4.
Molecules ; 26(7)2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33916405

RESUMO

The study aimed to investigate the antibacterial activity of Mustard (Brassica juncea) and Moringa (Moringa oleifera) leaf extracts and coagulant protein for their potential application in water treatment. Bacterial cell aggregation and growth kinetics studies were employed for thirteen bacterial strains with different concentrations of leaf extracts and coagulant protein. Moringa oleifera leaf extract (MOS) and coagulant protein showed cell aggregation against ten bacterial strains, whereas leaf extract alone showed growth inhibition of five bacterial strains for up to 6 h and five bacterial strains for up to 3 h. Brassica juncea leaf extract (BJS) showed growth inhibition for up to 6 h, and three bacterial strains showed inhibition for up to 3 h. The highest inhibition concentration with 2.5 mg/mL was 19 mm, and furthermore, the minimum inhibitory concentration (MIC) (0.5 mg/mL) and MBC (1.5 mg/mL) were determined to have a higher antibacterial effect for <3 KDa peptides. Based on LCMS analysis, napin was identified in both MOS and BJS; furthermore, the mode of action of napin peptide was determined on lipoprotein X complex (LpxC) and four-chained structured binding protein of bacterial type II topoisomerase (4PLB). The docking analysis has exhibited moderate to potent inhibition with a range of dock score -912.9 Kcal/mol. Thus, it possesses antibacterial-coagulant potential bioactive peptides present in the Moringa oleifera purified protein (MOP) and Brassica juncea purified protein (BJP) that could act as an effective antimicrobial agent to replace currently available antibiotics. The result implies that MOP and Brassica juncea purified coagulant (BJP) proteins may perform a wide degree of antibacterial functions against different pathogens.


Assuntos
Albuminas 2S de Plantas/química , Antibacterianos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Moringa oleifera/química , Mostardeira/química , Albuminas 2S de Plantas/isolamento & purificação , Albuminas 2S de Plantas/farmacologia , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Amidoidrolases/genética , Amidoidrolases/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Sítios de Ligação , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Folhas de Planta/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
5.
J Med Chem ; 64(7): 3997-4019, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33750129

RESUMO

Cardioprotective activity of dexrazoxane (ICRF-187), the only clinically approved drug against anthracycline-induced cardiotoxicity, has traditionally been attributed to its iron-chelating metabolite. However, recent experimental evidence suggested that the inhibition and/or depletion of topoisomerase IIß (TOP2B) by dexrazoxane could be cardioprotective. Hence, we evaluated a series of dexrazoxane analogues and found that their cardioprotective activity strongly correlated with their interaction with TOP2B in cardiomyocytes, but was independent of their iron chelation ability. Very tight structure-activity relationships were demonstrated on stereoisomeric forms of 4,4'-(butane-2,3-diyl)bis(piperazine-2,6-dione). In contrast to its rac-form 12, meso-derivative 11 (ICRF-193) showed a favorable binding mode to topoisomerase II in silico, inhibited and depleted TOP2B in cardiomyocytes more efficiently than dexrazoxane, and showed the highest cardioprotective efficiency. Importantly, the observed ICRF-193 cardioprotection did not interfere with the antiproliferative activity of anthracycline. Hence, this study identifies ICRF-193 as the new lead compound in the development of efficient cardioprotective agents.


Assuntos
Cardiotônicos/uso terapêutico , Cardiotoxicidade/tratamento farmacológico , Piperazinas/uso terapêutico , Inibidores da Topoisomerase II/uso terapêutico , Animais , Animais Recém-Nascidos , Cardiotônicos/síntese química , Cardiotônicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Daunorrubicina/toxicidade , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Miócitos Cardíacos/efeitos dos fármacos , Piperazinas/síntese química , Piperazinas/metabolismo , Ligação Proteica , Ratos Wistar , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/metabolismo
6.
Molecules ; 26(4)2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672694

RESUMO

A549 human lung carcinoma cell lines were treated with a series of new drugs with both tacrine and coumarin pharmacophores (derivatives 1a-2c) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. The ability of human topoisomerase I (hTOPI) and II to relax supercoiled plasmid DNA in the presence of various concentrations of the tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis. The biological activities of the derivatives were studied using MTT assays, clonogenic assays, cell cycle analysis and quantification of cell number and viability. The content and localization of the derivatives in the cells were analysed using flow cytometry and confocal microscopy. All of the studied compounds were found to have inhibited topoisomerase I activity completely. The effect of the tacrine-coumarin hybrid compounds on cancer cells is likely to be dependent on the length of the chain between the tacrine and coumarin moieties (1c, 1d = tacrine-(CH2)8-9-coumarin). The most active of the tested compounds, derivatives 1c and 1d, both display longer chains.


Assuntos
Antineoplásicos/farmacologia , Cumarínicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Tacrina/farmacologia , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/farmacologia , Células A549 , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cumarínicos/química , DNA Topoisomerases Tipo II/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Tacrina/química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase II/química , Células Tumorais Cultivadas
7.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672992

RESUMO

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.


Assuntos
Cromossomos de Plantas/genética , Hordeum/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Cromossomos de Plantas/química , Cromossomos de Plantas/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Corantes Fluorescentes/química , Hordeum/citologia , Indóis/química , Metáfase/genética , Reprodutibilidade dos Testes
8.
Phytomedicine ; 84: 153504, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33611211

RESUMO

BACKGROUND: DNA topoisomerase (Topo) inhibition plays key role in breast cancer treatment. Stephania hainanensis H. S. Lo et Y. Tsoong (S. hainanensis), a Li nationality plant that has abundant aporphine alkaloids, can inhibit Topo. PURPOSE: To identify a dual Topo inhibitor, a deep and systematic study of active aporphine alkaloids in S. hainanensis and their mechanisms of inhibiting breast cancer proliferation and Topo activity are essential. STUDY DESIGN: This study aimed to assess the anti-breast cancer and Topo inhibitory activities of oxocrebanine and explore the underlying mechanisms. METHODS: The growth inhibitory activities of 12 compounds in S. hainanensis were screened by MTT assay in MCF-7, SGC-7901, HepG-2 cells, and compared with the effects on human normal mammary epithelial MCF-10A cells as non cancer control cells. The Topo inhibitory activity was assessed by DNA relaxation and unwinding assays, kDNA decatenation assay and western blot. Cell cycle and autophagy analyses were carried out with flow cytometry and staining. Acridine orange staining and α-tubulin morphology were observed by fluorescence microscopy. Western blot was used to examine microtubule assembly dynamics and the expression levels of key proteins associated with DNA damage, autophagy and mitotic arrest. RESULTS: Oxocrebanine was the anti-breast cancer active alkaloid in S. hainanensis. It exhibited the best inhibitory effect on MCF-7 cells with an IC50 of 16.66 µmol/l, and had only weak effect on the proliferation of MCF-10A cells. Oxocrebanine inhibited Topo I and II α in a cell-free system and in MCF-7 cells. The DNA unwinding assay suggested that oxocrebanine intercalated with DNA as a catalytic inhibitor. Oxocrebanine regulated the levels of Topo I and IIα and DNA damage-related proteins. Oxocrebanine led to the mitotic arrest, and these effects occurred through both p53-dependent and p53-independent pathways. Oxocrebanine induced autophagy, abnormal α-tubulin morphology and stimulated enhanced microtubule dynamics. CONCLUSION: Oxocrebanine was the anti-breast cancer active aporphine alkaloid in S. hainanensis. Oxocrebanine was a Topo I/IIα dual inhibitor, catalytic inhibitor and DNA intercalator. Oxocrebanine caused DNA damage, autophagy, and mitotic arrest in MCF-7 cells. Oxocrebanine also disrupted tubulin polymerization. Accordingly, oxocrebanine held a great potential for development as a novel dual Topo inhibitor for effective breast cancer treatment.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Inibidores da Topoisomerase/farmacologia , Alcaloides/farmacologia , Antineoplásicos Fitogênicos/química , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Células MCF-7 , Mitose/efeitos dos fármacos , Inibidores da Topoisomerase/química
9.
PLoS Comput Biol ; 17(1): e1007814, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465072

RESUMO

DNA topoisomerase II-ß (TOP2B) is fundamental to remove topological problems linked to DNA metabolism and 3D chromatin architecture, but its cut-and-reseal catalytic mechanism can accidentally cause DNA double-strand breaks (DSBs) that can seriously compromise genome integrity. Understanding the factors that determine the genome-wide distribution of TOP2B is therefore not only essential for a complete knowledge of genome dynamics and organization, but also for the implications of TOP2-induced DSBs in the origin of oncogenic translocations and other types of chromosomal rearrangements. Here, we conduct a machine-learning approach for the prediction of TOP2B binding using publicly available sequencing data. We achieve highly accurate predictions, with accessible chromatin and architectural factors being the most informative features. Strikingly, TOP2B is sufficiently explained by only three features: DNase I hypersensitivity, CTCF and cohesin binding, for which genome-wide data are widely available. Based on this, we develop a predictive model for TOP2B genome-wide binding that can be used across cell lines and species, and generate virtual probability tracks that accurately mirror experimental ChIP-seq data. Our results deepen our knowledge on how the accessibility and 3D organization of chromatin determine TOP2B function, and constitute a proof of principle regarding the in silico prediction of sequence-independent chromatin-binding factors.


Assuntos
Cromatina , DNA Topoisomerases Tipo II , Genoma/genética , Modelos Genéticos , Animais , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Genômica , Humanos , Células MCF-7 , Aprendizado de Máquina , Camundongos , Ligação Proteica , Timócitos
10.
Nat Struct Mol Biol ; 28(1): 92-102, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398171

RESUMO

Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex-duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.


Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Meiose/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Microscopia de Força Atômica , Mutação/genética , Conformação de Ácido Nucleico , Recombinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Chemistry ; 27(20): 6254-6262, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33465263

RESUMO

Two series of the ferrocenyl and ruthenocenyl analogues of etoposide bearing 1,2,3-triazolyl or aminoalkyl linker were synthesized and evaluated for their cytotoxic properties, influence on the cell cycle, ability to induce tubulin polymerization, and inhibition of topoisomerase II activity. We found that the replacement of the etoposide carbohydrate moiety with a metallocenyl group led to organometallic conjugates exhibiting differentiated antiproliferative activity. Biological studies demonstrated that two ferrocenylalkylamino conjugates were notably more active than etoposide, with submicromolar or low-micromolar IC50 values towards SW620, etoposide-resistant SW620E, and methotrexate-resistant SW620M cancer cell lines. Moreover, the simplest ferrocenylmethylamino conjugate exerted dual inhibitory action against tubulin polymerization and topoisomerase II activity while other studied compounds affected only topoisomerase II activity.


Assuntos
Antineoplásicos , Tubulina (Proteína) , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo II/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Etoposídeo/farmacologia , Polimerização , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/farmacologia , Tubulina (Proteína)/metabolismo
12.
Int J Biol Macromol ; 170: 523-531, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33387542

RESUMO

Precise monitoring of the enzyme activity by a suitable modulator is one of the very fundamental aspects of drug designing that provides the opportunity to overcome the challenges of several diseases. Herein, inhibition of human Topoisomerase IIα enzyme which serves as a potential target site for several anti-cancer drugs is demonstrated by using ultra-small size gold nanoclusters (Au NCs) with the dimension comparable with size of the active site of the enzyme. Molecular dynamics simulation results demonstrate that the Au NCs strongly interact with the human Topo IIα enzyme at its active site or allosteric site depending on forms of enzyme. Additionally, binding energy and interaction profile provides the molecular basis of understanding of interactions of ultra-small size Au NCs and human Topo IIα enzyme. Enthalpy change (ΔH) and binding constant (K) are measured based on a sequential binding model of the Au NCs with the enzyme as demonstrated by the ITC study. Moreover, the in-vitro inhibition study of the catalytic activity of the enzyme and gel electrophoresis indicates that the ultra-small size Au NCs may be used as a potent inhibitor of human Topo IIα enzyme.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Corantes Fluorescentes/química , Ouro/química , Nanopartículas Metálicas/química , Sítio Alostérico/efeitos dos fármacos , Catálise/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , DNA Topoisomerases Tipo II/química , Humanos , Simulação de Dinâmica Molecular , Neoplasias/metabolismo
13.
BMC Cancer ; 21(1): 39, 2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33413211

RESUMO

BACKGROUND: To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer. METHODS: We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit. RESULTS: We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%). CONCLUSIONS: We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample.


Assuntos
Ciclina E/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Biópsia Líquida/métodos , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Lesões Pré-Cancerosas/patologia , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasia Intraepitelial Cervical/metabolismo , Neoplasia Intraepitelial Cervical/patologia , Neoplasia Intraepitelial Cervical/cirurgia , Neoplasia Intraepitelial Cervical/virologia , Citodiagnóstico/métodos , Progressão da Doença , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/cirurgia , Lesões Pré-Cancerosas/virologia , Prognóstico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/cirurgia , Neoplasias do Colo do Útero/virologia
14.
Mol Med Rep ; 23(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33355372

RESUMO

Neochlorogenic acid (NCA), a natural compound found in honeysuckle, possesses prominent anti­inflammatory and antitumor effects. Pingyangmycin (PYM) induces DNA damage and has been used for the treatment of oral and maxillofacial tumors. Oral care serves an important role in promoting wound healing during chemotherapy in patients with oral squamous cell carcinoma (OSCC). Therefore, the present study aimed to analyze the effects of NCA and PYM on OSCC cells and to investigate the potential underlying mechanism. Reverse transcription­quantitative PCR and western blotting were conducted to analyze the expression levels of DNA topoisomerase II α (TOP2A) in different OSCC cell lines. TOP2A­overexpression cells were constructed via transfection of TOP2A­overexpression plasmids. Following NCA or PYM treatment, cell proliferation was assessed using Cell Counting Kit­8 and colony formation assays, whereas cell apoptosis and the cell cycle distribution were assessed via TUNEL staining and flow cytometry, respectively. In addition, the expression levels of apoptosis­ and cell cycle­related proteins were detected via western blotting. Moreover, co­immunoprecipitation (Co­IP) was conducted to determine whether TOP2A interacted with CDK1. The results of the present study indicated that NCA treatment significantly enhanced the suppressive effects of PYM on OSCC cell proliferation and apoptosis. The results also indicated that PYM arrested the cell cycle in the G0/1 by regulating cyclin dependent kinase 1 (CDK1)/cyclin B1, which was enhanced by the cotreatment of NCA and PYM. In addition, NCA and PYA treatment altered the expression levels of apoptosis­related proteins. The Co­IP assay indicated that TOP2A interacted with CDK1. Moreover, TOP2A overexpression significantly reversed the effects of NCA and PYM treatment on OSCC cell proliferation and apoptosis. In addition, NCA significantly decreased PYM­induced toxicity in normal oral epithelial cells. In conclusion, the results of the present study suggested that NCA may promote the inhibitory effects of PYM in OSCC via TOP2A.


Assuntos
Antineoplásicos/farmacologia , Bleomicina/análogos & derivados , Ácido Clorogênico/análogos & derivados , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ácido Quínico/análogos & derivados , Bleomicina/agonistas , Bleomicina/farmacologia , Linhagem Celular Tumoral , Ácido Clorogênico/agonistas , Ácido Clorogênico/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Ácido Quínico/agonistas , Ácido Quínico/farmacologia
15.
Med Sci Monit ; 26: e929120, 2020 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-33361736

RESUMO

BACKGROUND This study was carried out to analyze TOP2A expression in lung adenocarcinoma (LUAD) and to assess its value in clinical diagnosis and prognosis. MATERIAL AND METHODS The Cancer Genome Atlas (TCGA) database was used to study the relationship of TOP2A expression with the progression and prognosis of LUAD. For a further elucidation of the value of TOP2A in LUAD, the effect of TOP2A knockout on cell viability and related protein expression of LUAD cell line A549 in vitro was investigated by using RNA interference, MTT, flow cytometry, RT-PCR, and western blot analysis. RESULTS According to the results of database analysis, TOP2A expression in LUAD was higher than that in normal lung tissues. There was a strong correlation of TOP2A expression with clinicopathological and epidemiological parameters of LUAD. The survival rate of LUAD patients with high TOP2A expression was lower than that of patients with low expression (P<0.001). The expression of TOP2A in A549 cells was higher than that in Beas-2B cells. After decreased expression of TOP2A in A549 cells, the proliferation of A549 cells was downregulated and the apoptosis rate was increased. It was further verified that TOP2A low expression exerts a role in LUAD through activation of the ERK/JNK/p-P38/CHOP signaling pathway. CONCLUSIONS The findings from this study showed that TOP2A expression was upregulated in a human lung adenocarcinoma cell line, and this finding was supported by bioinformatics analysis. Further studies are required to determine whether TOP2A expression is a prognostic biomarker and potential therapeutic target in patients with lung adenocarcinoma.


Assuntos
Adenocarcinoma de Pulmão/genética , DNA Topoisomerases Tipo II/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Células A549 , Adenocarcinoma de Pulmão/patologia , DNA Topoisomerases Tipo II/metabolismo , Regulação para Baixo/genética , Estudos de Associação Genética , Humanos , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do Tratamento
16.
PLoS One ; 15(9): e0239466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32960919

RESUMO

DNA topoisomerase II (topo II) is an essential enzyme that regulates DNA topology by DNA cleavage and re-ligation. In vertebrates, there are two isozymes, α and ß. The C-terminal domain (CTD) of the isozymes, which shows a low degree of sequence homology between α and ß, is involved in each isozyme-specific intracellular behavior. The CTD of topo IIß is supposedly involved in topo II regulation. Topo IIß is maintained in an inactive state in the nucleoli by the binding of RNA to the 50-residue region termed C-terminal regulatory domain (CRD) present in the CTD. Although in vitro biochemical analysis indicates that the CTD of topo IIß has DNA binding activity, it is unclear whether CTD influences catalytic reaction in the nucleoplasm. Here, we show that the proximal CTD (hereafter referred to as pCTD) of rat topo IIß, including the CRD, is involved in the catalytic reaction in the nucleoplasm. We identified the pCTD as a domain with DNA binding activity by in vitro catenation assay and electrophoretic mobility shift assay. Fluorescence recovery after photo-bleaching (FRAP) analysis of pCTD-lacking mutant (ΔpCTD) showed higher mobility in nucleoplasm than that of the wild-type enzyme, indicating that the pCTD also affected the nuclear dynamics of topo IIß. ICRF-193, one of the topo II catalytic inhibitors, induces the formation of closed-clamp intermediates of topo II. Treatment of ΔpCTD with ICRF-193 significantly decreased the efficiency of closed-clamp formation. Altogether, our data indicate that the binding of topo IIß to DNA through the pCTD is required for the catalytic reaction in the nucleoplasm.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Piperazinas/farmacologia , Animais , Catálise/efeitos dos fármacos , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , DNA/metabolismo , Células HEK293 , Humanos , RNA/metabolismo , Ratos
17.
Oncogene ; 39(27): 5068-5081, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32528131

RESUMO

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We and others had shown that a subset of glioblastomas, the most malignant of all primary brain tumors in adults, is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, and 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 led to resistance to etoposide and doxorubicin and impaired the induction of proapoptotic gene APAF1 following treatment. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from gliomas and multiple other cancers.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Etoposídeo/farmacologia , Glioblastoma/tratamento farmacológico , Proteínas Ribossômicas/metabolismo , Inibidores da Topoisomerase II/farmacologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Neoplasias Encefálicas/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Doxorrubicina/farmacologia , Técnicas de Inativação de Genes , Glioblastoma/genética , Humanos
18.
Mol Pharmacol ; 98(3): 222-233, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32587095

RESUMO

DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Nucleosídeos/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Sulfonamidas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Linhagem Celular , DNA/metabolismo , DNA Topoisomerases Tipo II/química , Humanos , Células K562 , Proteínas de Ligação a Poli-ADP-Ribose/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
19.
Gene ; 754: 144859, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32535049

RESUMO

DNA topoisomerases essentially remove topological strains generated during DNA replication, transcription, DNA repair, and other cytogenetic processes. However, distinct expression level and prognostic significance of individual topoisomerase isoforms in gastric cancer (GC) remain largely unexplored. In this study, we utilized Oncomine and Kaplan-Meier plotter database to detect the mRNA expression level of individual topoisomerase isoforms as well as assess their prognostic significance in GC patients. With the exception of TOP3B and TOP2B, levels of all topoisomerase isoforms were found to be elevated in GC patients when compared to the normal tissues. Elevated expression of TOP1 and TOP1MT was relevant to longer overall survival (OS) in GC and gastric intestinal type adenocarcinoma (GITA) patients, but not in diffuse gastric adenocarcinoma (DFA) patients. Increased expression of TOP2A and TOP2B was related to better OS in GC, as well as in GITA and DFA patients. In contrast, increased expression TOP3A and TOP3B was associated with shorter OS in GC, as well as in GITA and DFA patients. We also applied the Tumor IMmune Estimation Resource (TIMER) tool to assess the correlations between distinct topoisomerase isoforms and the infiltrating immune cell landscape. Furthermore, we found that down-regulating the expression of TOP3A by shRNA significantly inhibited the proliferation and colony formation in GC cells compared to control shRNA treated cells. Thus our study lays the framework for utilizing topoisomerases in better understanding the complexity and heterogeneity of GC and for developing strategies for novel customized therapy in GC patients.


Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias Gástricas/patologia , Adenocarcinoma/enzimologia , Biomarcadores Tumorais/genética , DNA Topoisomerases Tipo II/genética , Precursores Enzimáticos , Perfilação da Expressão Gênica , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Prognóstico , Neoplasias Gástricas/enzimologia , Taxa de Sobrevida
20.
Sci Rep ; 10(1): 8846, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483162

RESUMO

Rare or orphan diseases affect only small populations, thereby limiting the economic incentive for the drug development process, often resulting in a lack of progress towards treatment. Drug repositioning is a promising approach in these cases, due to its low cost. In this approach, one attempts to identify new purposes for existing drugs that have already been developed and approved for use. By applying the process of drug repositioning to identify novel treatments for rare diseases, we can overcome the lack of economic incentives and make concrete progress towards new therapies. Adrenocortical Carcinoma (ACC) is a rare disease with no practical and definitive therapeutic approach. We apply Heter-LP, a new method of drug repositioning, to suggest novel therapeutic avenues for ACC. Our analysis identifies innovative putative drug-disease, drug-target, and disease-target relationships for ACC, which include Cosyntropin (drug) and DHCR7, IGF1R, MC1R, MAP3K3, TOP2A (protein targets). When results are analyzed using all available information, a number of novel predicted associations related to ACC appear to be valid according to current knowledge. We expect the predicted relations will be useful for drug repositioning in ACC since the resulting ranked lists of drugs and protein targets can be used to expedite the necessary clinical processes.


Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Reposicionamento de Medicamentos/métodos , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/patologia , Biologia Computacional , Cosintropina/uso terapêutico , DNA Topoisomerases Tipo II/metabolismo , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo
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