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This study evaluated the genotoxicity of Ethanol Extract (EEEp), Dichloromethane Fraction (FDCMEp) and isoeleutherin isolated from Eleutherine plicata, using the micronucleus test and the impact of structural alterations on toxicity and molecular docking (topoisomerase II and DNA complex). The extract was obtained by maceration and fractionation in a chromatography column. The genotoxicity was evaluated by the micronucleus test in human hepatoma cells (HepG2). Isoeleutherin was the starting molecule in the search for analogues by structural similarity, using the ZINC and e-Molecules databases. Isoeleutherin and analogues were subjected to in silico toxicity prediction, and compounds free of toxicological risks (CP13, CP14, CP17 and isoeleutherin) were selected for molecular docking in Topoisomerase II (PDB: 1ZXM). In the micronucleus test, isoeleutherin was less genotoxic. Among the 22 isoeleutherin analogues there were variations in the toxicity profile. Molecular docking studies showed that the compounds have good complementarity in the active site with important hydrogens bonds. Therefore, the structural changes of isoeleutherin led to the obtaining of a molecule with a lower mutagenic potential, and the CP13 can be considered a prototype compound for the development of new molecules with pharmacological potential.
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Dano ao DNA , DNA Topoisomerases Tipo II , Humanos , Simulação de Acoplamento Molecular , Caspase 8RESUMO
Introduction: Epidemiological studies around the world on acute leukemia (AL) and risk factors in infants are scarce. Infant AL has been proposed to originate in utero, which facilitates its study by establishing a short exposure time in pregnant women to environmental and dietary factors that could contribute to the risk of or protection against leukemia. We hypothesized that maternal diet during pregnancy may be an important factor involved in AL in offspring. Methods: We conducted a hospital-based case-control study from 2010 to 2019 on maternal diet during pregnancy in nine high-specialty public hospitals of different health institutions that diagnose and offer treatment to children with AL in Mexico City. Cases (n=109) were children ≤24 months of age with de novo diagnosis of AL, and controls (n=252) were children obtained in hospitals from second-level medical care matched for age, sex, and health institution. Maternal diet during pregnancy was obtained by a semiquantitative food frequency questionnaire. Unconditional logistic regression models were used to assess the association between food groups and infant AL. Potential confounders were assessed by constructing directed acyclic graphs (DAGs) with Dagitty software in which adjusted options were identified for the construction of unconditional logistic regression models. Results: Cases were slightly predominantly female (52.3%). The years of education of the mother in cases and controls was 0-9 on average, and those who reported smoking cigarettes and consuming alcohol during pregnancy did so at a low frequency. Regarding the mother's diet, the main findings were that the consumption of allium vegetables during pregnancy was inversely associated with AL for medium and high consumption (OR=0.26, 95% CI 0.14-0.46; P-trend< 0.001). In contrast, the high consumption of high-fat dairy products had a positive association with AL (OR=2.37, 95% CI 1.30-4.34; P-trend<0.001). No association was found between consumption of topoisomerase II inhibitor foods during pregnancy and AL. Conclusion: The results suggest that maternal intake during pregnancy of allium vegetables, specifically garlic, is inversely associated with the development of AL in children ≤24 months old. On the other hand, consumption of high-fat dairy products is positively associated with AL in children ≤24 months old.
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In this study, we report the synthesis of twenty new acridine-thiosemicarbazone derivatives and their antiproliferative activities. Mechanisms of action such as the inhibition of topoisomerase IIα and the interaction with DNA have been studied for some of the most active derivatives by means of both in silico and in vitro methods, and evaluations of the non-clinical toxicities (in vivo) in mice. In general, the compounds showed greater cytotoxicity against B16-F10 cells, with the highest potency for DL-08 (IC50 = 14.79 µM). Derivatives DL-01 (77%), DL-07 (74%) and DL-08 (79%) showed interesting inhibition of topoisomerase IIα when compared to amsacrine, at 100 µM. In silico studies proposed the way of bonding of these compounds and a possible stereoelectronic reason for the absence of enzymatic activity for CL-07 and DL-06. Interactions with DNA presented different spectroscopic effects and indicate that the compound CL-07 has higher affinity for DNA (Kb = 4.75 × 104 M-1; Ksv = 2.6 × 103 M-1). In addition, compounds selected for non-clinical toxicity testing did not show serious signs of toxicity at the dose of 2000 mg/kg in mice; cytotoxic tests performed on leukemic cells (K-562) and its resistant form (K-562 Lucena 1) identified moderate potency for DL-01 and DL-08, with IC50 between 11.45 and 17.32 µM.
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Topoisomerase inhibitors are extensively used in cancer chemotherapy. In the process of identifying novel anticancer compounds, biological evaluations are crucial and include, among others, the use of in silico and in vitro approaches. This work aimed to present recent research involving the obtainment and in silico and in vitro evaluation of topoisomerase I, II, and double inhibitors, of synthetic and natural origin, as potential compounds against tumor cells, in addition to proposing the construction of a desirable enzyme catalytic site. Therefore, it was observed that most Topoisomerase I inhibitors presented medium to large structures, with a rigid portion and a flexible region. In contrast, Topoisomerase IIα inhibitors showed medium and large structural characteristics, in addition to the planarity of the aromatic rings, which are mitigated due to flexible rings but may also present elements that restrict conformation. Most compounds that exhibit dual inhibitory activity had relatively long chains, in addition to a flat and rigid portion suggestive of affinity for Topo I and a flexible region characteristic of selective drugs for Topo II. Besides, it is noticed that most compounds that exhibit dual inhibitory showed similarities in the types of interactions and amino acids when compared to the selective compounds of Topo I and II. For instance, selective Topoisomerase I inhibitors interact with Arginine364 residues, and selective Topoisomerase II inhibitors interact with Arginine487 residues, as both residues are targets for dual compounds.
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Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/química , Proliferação de Células , DNA Topoisomerases Tipo II/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Neoplasias/tratamento farmacológico , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase I/uso terapêutico , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/metabolismoRESUMO
DNA topoisomerase II enzymes maintain DNA stability during vital processes, such as genome replication, transcription and chromosomal segregation during mitosis and meiosis. In the present work, we analyzed functional aspects of the DNA topoisomerase II (AeTopII) enzyme of the mosquito Aedes aegypti. Here, we show that AeTopII mRNA is expressed at all stages of mosquito development. By in situ hybridization, we found that the AeTopII mRNA is concentrated along the ovarian follicular cells as well as in the region of the follicles. The observed expression profiles likely reflect increased topoisomerase II cellular requirements due to the intense ovarian growth and egg production following blood feeding in Ae. aegypti females. The drug etoposide, a classic inhibitor of topoisomerase II, was used for in vivo testing with 2nd stage larvae, in order to investigate the functional importance of this enzyme in Ae. aegypti survival and development. Inhibition of topoisomerase II activity with etoposide concentrations ranging from 10 to 200 µM did not leads to the immediate death of larvae. However, after 10 days of observation, etoposide treatments resulted in 30-40% decrease in survival, in a dose dependent manner, with persisting larvae and pupae presenting incomplete development, as well as morphological abnormalities. Also, approximately 50% of the treated larvae did not reach the pupal stage. Thus, we conclude that AeTopII is a vital enzyme in the development of Ae. aegypti and its sensitivity to inhibitors should be explored for potential chemical agents to be used in vector control.
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Aedes , DNA Topoisomerases Tipo II/metabolismo , Etoposídeo/toxicidade , Larva/efeitos dos fármacos , Mosquitos Vetores/efeitos dos fármacos , Inibidores da Topoisomerase II/toxicidade , Aedes/enzimologia , Aedes/crescimento & desenvolvimento , AnimaisRESUMO
Prostaglandin A2-AcMe (1) and Prostaglandin A2 (2) were isolated from the octocoral Plexaura homomalla and three semisynthetic derivatives (3-5) were then obtained using a reduction protocol. All compounds were identified through one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) experiments. Additionally, evaluation of in vitro cytotoxic activity against the breast (MDA-MB-213) and lung (A549) cancer cell lines, in combination with enzymatic activity and molecular docking studies with the enzymes p38α-kinase, Src-kinase, and topoisomerase IIα, were carried out for compounds 1-5 in order to explore their potential as inhibitors of cancer-related molecular targets. Results showed that prostaglandin A2 (2) was the most potent compound with an IC50 of 16.46 and 25.20 µg/mL against MDA-MB-213 and A549 cell lines, respectively. In addition, this compound also inhibited p38α-kinase in 49% and Src-kinase in 59% at 2.5 µM, whereas topoisomerase IIα was inhibited in 64% at 10 µM. Enzymatic activity was found to be consistent with molecular docking simulations, since compound 2 also showed the lowest docking scores against the topoisomerase IIα and Src-kinase (-8.7 and -8.9 kcal/mol, respectively). Thus, molecular docking led to establish some insights into the predicted binding modes. Results suggest that prostaglandin 2 can be considered as a potential lead for development inhibitors against some enzymes present in cancer processes.
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Antozoários , Antineoplásicos/farmacologia , Prostaglandinas/farmacologia , Células A549/efeitos dos fármacos , Animais , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Oceanos e MaresRESUMO
In the present study, acridine-thiosemicarbazones (ATD) derivatives were tested for their interaction properties with BSA through UV-Vis absorption and fluorescence spectroscopic studies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated after the derivatives were added to the BSA. Values for the binding constant (Kb) ranged from 1.62â¯×â¯104 to 8.71â¯×â¯105â¯M-1 and quenching constant (KSV) from 3.46â¯×â¯102 to 7.83â¯×â¯103â¯M-1 indicating a good affinity to BSA protein. Complementary, two compounds were selected to assess their inhibition activity against topoisomerase IIα enzyme, of which derivative 3a presented the best result. Moreover, to evaluate protein-ligand interactions, as well as the antitopoisomerase potential of these compounds, tests of molecular modeling were performed between all compounds using the albumin and Topoisomerase IIα/DNA complex. Finally, in silico studies showed that all derivatives used in this research displayed good oral bioavailability potential.
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Acridinas/química , Soroalbumina Bovina/química , Tiossemicarbazonas/química , Inibidores da Topoisomerase/química , Inibidores da Topoisomerase/farmacologia , Técnicas de Química Sintética , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Soroalbumina Bovina/metabolismo , Análise Espectral , Relação Estrutura-Atividade , Inibidores da Topoisomerase/síntese química , Inibidores da Topoisomerase/metabolismoRESUMO
Nine new spiroacridine derivatives were synthetized by introducing cyano-N-acylhydrazone group between the acridine and phenyl-substituted rings followed by spontaneous cyclization. The new compounds were assayed for their DNA binding properties, human topoisomerase IIα inhibition and bovine serum albumin (BSA) interaction. Besides, docking analysis were performed in order to better understanding the biomolecule-compounds interactions. All compounds interacted with BSA which was demonstrated by the fluorescence suppression constant of 104â¯M-1. Compounds with chloro and NO2 substituents at that para-position on phenyl ring demonstrated the best results for BSA interaction. DNA binding constant determined by UV-vis data demonstrated high values for AMTAC-11 and AMTAC-14, 1.1â¯×â¯108â¯M-1 and 4.8â¯×â¯106â¯M-1, respectively, and all others presented constant values of 105â¯M-1. AMTAC-06 with chloro at para-position on phenyl ring presented a topoisomerase II inhibition of 84.34% in comparison to the positive controls used. Docking studies indicated that AMTAC-06 is able to intercalate the DNA base pairs at topoisomerase IIα active site, preventing DNA connection after break, in a process known as poisoning. Topoisomerase enzyme inhibition result was correlated to BSA interaction profile, since AMTAC-06 showed the best results in both analysis. The findings obtained here proved that methoxy or chloro substitution on phenyl ring at para-position is fundamental for in vitro activity of new spiroacridine derivatives, and indicates that AMTAC-06 is a promising entity and should serve as a lead compound in the development of new DNA and protein binders, as well as human topoisomerase II inhibitors.
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Acridinas/farmacologia , DNA Topoisomerases Tipo II/metabolismo , DNA/química , Soroalbumina Bovina/química , Inibidores da Topoisomerase II/farmacologia , Acridinas/síntese química , Acridinas/química , Animais , Bovinos , Relação Dose-Resposta a Droga , Fluorescência , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/químicaRESUMO
The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanism of Top2 poisons involves a drug-mediated stabilization of a Top2-DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5' end of DNA covalently bound to a tyrosine from Top2 through a phosphodiester group. Drug-stabilized Top2cc leads to Top2-linked-DNA breaks, which are believed to mediate their cytotoxicity. Several time-consuming or cell type-limiting assays have been used in the past to study drug-stabilized Top2cc. Here, we describe a flow cytometry-based method that allows a rapid assessment of drug-induced Top2cc, which is suitable for high throughput analysis in almost any kind of human cell. The analyses of the drug-induced Top2cc in the cell cycle context and the possibility to track its removal are additional benefits from this methodology. © 2017 by John Wiley & Sons, Inc.
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DNA Topoisomerases Tipo II/análise , DNA/análise , Etoposídeo/química , Citometria de Fluxo/métodos , Animais , DNA/química , DNA/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Etoposídeo/farmacologia , Células HL-60 , HumanosRESUMO
The present study aimed to ascertain the significance of topoisomerase II α (TOP2A) and minichromosome maintenance protein (MCM) 2 expression in anal carcinoma. A total of 75 anal lesions were retrieved from the files of the Department of Pathology of Barretos Cancer Hospital (Barretos, Brazil) in order to verify the human papillomavirus (HPV) statuses of these lesions and characterize the immunohistochemical expression levels of TOP2A and MCM2 in anal carcinoma, as these are important markers for cervical HPV-induced lesions; their expression was also compared with respect to p16 and Ki-67. The vast majority of the cases tested positive for HPV16 (84%); 1 case tested positive for both HPV16 and HPV18. Positive HPV16 status was more frequent in early stages than in advanced stages (P=0.008). Positive immunohistochemical reactivity for MCM2 and TOP2A protein was observed in 71.6 and 100% of cases, respectively. Positive reactivity for p16 was significantly associated (P=0.001) with histological grade, and was more commonly expressed in squamous cell carcinoma than adenocarcinomas. HPV16 was strongly associated with positive p16 protein expression (76.6%). However, the high expression of Ki-67 combined with the high expression of p16 was predominantly observed in Stage III-IV cases. MCM2, TOP2A, p16 and Ki-67 exhibited intense positive staining in the anal lesions, indicating that these markers were significantly and constantly expressed in anal carcinoma.
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Topoisomerase II (Top2) is an important target for anticancer therapy. A variety of drugs that poison Top2, including several epipodophyllotoxins, anthracyclines, and anthracenediones, are widely used in the clinic for both hematologic and solid tumors. The poisoning of Top2 involves the formation of a reaction intermediate Top2-DNA, termed Top2 cleavage complex (Top2cc), which is persistent in the presence of the drug and involves a 5' end of DNA covalently bound to a tyrosine from the enzyme through a phosphodiester group. Drug-induced Top2cc leads to Top2 linked-DNA breaks which are the major responsible for their cytotoxicity. While biochemical detection is very laborious, quantification of drug-induced Top2cc by immunofluorescence-based microscopy techniques is time consuming and requires extensive image segmentation for the analysis of a small population of cells. Here, we developed a flow cytometry-based method for the analysis of drug-induced Top2cc. This method allows a rapid analysis of a high number of cells in their cell cycle phase context. Moreover, it can be applied to almost any human cell type, including clinical samples. The methodology is useful for a high-throughput analysis of drugs that poison Top2, allowing not just the discrimination of the Top2 isoform that is targeted but also to track its removal. © 2016 International Society for Advancement of Cytometry.
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DNA Topoisomerases Tipo II/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Citometria de Fluxo/métodos , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase II/química , Antraquinonas/química , Antraquinonas/isolamento & purificação , Antraquinonas/uso terapêutico , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Triagem em Larga Escala , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/isolamento & purificação , Inibidores da Topoisomerase II/isolamento & purificação , Inibidores da Topoisomerase II/uso terapêuticoRESUMO
In cancer treatment, chemotherapy is one of the main strategies used. The knowledge of the cellular and molecular characteristics of tumors allows the use of more specific drugs, making the removal of tumors more efficient. Among the drugs of choice in these treatments, topoisomerase inhibitors are widely used against different types of tumors. Topoisomerases are enzymes responsible for maintaining the structure of DNA, altering its topological state temporarily during the processes of replication and transcription, in order to avoid supercoiling and entanglements at the double helix. The DNA damage formed as a result of topoisomerase inhibition can be repaired by DNA repair mechanisms. Thus, DNA repair pathways can modulate the effectiveness of chemotherapy. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the main pathways involved in the removal of double strand breaks (DSBs); while nucleotide excision repair (NER) is mainly characterized by the removal of lesions that lead to significant structural distortions in the DNA double helix. Evidence has shown that DSBs are the main type of damage resulting from the inhibition of the DNA topoisomerase II enzyme, and therefore the involvement of HR and NHEJ pathways in the repair process is well established. However, some topoisomerase II inhibitors induce other types of lesions, like DNA adducts, interstrand crosslinks and reactive oxygen species, and studies have shown that other DNA repair pathways might be participating in removing injury induced by these drugs. This review aims to correlate the involvement of proteins from different DNA repair pathways in response to these drugs, with an emphasis on NER.
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Dano ao DNA/efeitos dos fármacos , Reparo do DNA , DNA Topoisomerases/metabolismo , Inibidores da Topoisomerase II/farmacologia , Animais , Adutos de DNA , Humanos , Oxirredução , Espécies Reativas de Oxigênio , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/químicaRESUMO
Mitoxantrone (MXT) is an anticancer drug structurally related to anthracyclines, such as doxorubicin (DOX). Here we report that cells deficient in nucleotide excision repair (NER) are very sensitive to MXT. However, cells deficient in each of the NER sub-pathways - transcription coupled repair (deficient in CSB protein) and global genome repair (deficient in XPC protein) - demonstrate a difference in sensitivity from each other and also show different responses in cell cycle profile, DNA synthesis and topo II DNA complex formation upon MXT treatment. XPC-deficient cells are slightly more resistant than CSB-deficient cells, and in the same way as MRC5 NER-proficient cells, show G2/M arrest, normal DNA synthesis rate and a pattern of formation of complexes similar to proficient cells, whereas CSB-deficient cells show accumulation in S phase, reduced DNA synthesis and a more intense signal of topo II DNA complexes, indicating that they remain longer in these cells. Complementation of CSB mutant cells with CSB rescue MXT-induced sensitivity and also a decrease in the signal intensity of the complexes, suggest that resolution of these lesions would take place. Taken together, our results indicate that NER proteins are implicated in the response to MXT and that CSB protein has a key role in processing MXT-induced topo II DNA complexes.
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Antineoplásicos/farmacologia , Reparo do DNA/efeitos dos fármacos , Mitoxantrona/farmacologia , Inibidores da Topoisomerase II/farmacologia , DNA/biossíntese , DNA/química , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a DNA repair enzyme that removes irreversible protein-linked 3' DNA complexes, 3' phosphoglycolates, alkylation damage-induced DNA breaks, and 3' deoxyribose nucleosides. In addition to its extended spectrum of substrates, TDP1 interacts with several DNA damage response factors. To determine whether TDP1 participates in the repair of topoisomerase II (Top2) induced DNA lesions, we generated TDP1 depleted (TDP1kd) human tumoral cells. We found that TDP1kd cells are hypersensitive to etoposide (ETO). Moreover, we established in a chromatin context that following treatment with ETO, TDP1kd cells accumulate increased amounts of Top2α cleavage complexes, removing them with an altered kinetics. We also showed that TDP1 depleted cells accumulate increased γH2AX and pS296Chk1 signals following treatment with ETO. Similarly, cytogenetics analyses following Top2 poisoning revealed increased amounts of chromatid and chromosome breaks and exchanges on TDP1kd cells in the presence or not of the DNA-PKcs inhibitor NU7026. However, the levels of sister chromatid exchanges were similar in both TDP1kd and control non-silenced cell lines. This suggests a role of TDP1 in both canonical non-homologous end joining and alternative end joining, but not in the homologous recombination repair pathway. Finally, micronucleus analyses following ETO treatment revealed a higher frequency of micronucleus containing γH2AX signals on TDP1kd cells. Together, our results highlight an active role of TDP1 in the repair of Top2-induced DNA damage and its relevance on the genome stability maintenance in human cells.
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Antígenos de Neoplasias/toxicidade , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Topoisomerases Tipo II/toxicidade , Proteínas de Ligação a DNA/toxicidade , Diester Fosfórico Hidrolases/metabolismo , Cromonas , Ensaio de Unidades Formadoras de Colônias , Reparo do DNA por Junção de Extremidades/fisiologia , Primers do DNA/genética , Etoposídeo/farmacologia , Citometria de Fluxo , Imunofluorescência , Violeta Genciana , Células HeLa , Histonas/metabolismo , Humanos , Immunoblotting , Testes para Micronúcleos , Morfolinas , Diester Fosfórico Hidrolases/deficiência , Diester Fosfórico Hidrolases/genética , Proteínas de Ligação a Poli-ADP-Ribose , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Etoposide (ETO), a drug used for the treatment of human tumors, is associated with the development of secondary malignancies. Recently, therapeutic strategies have incorporated chemosensitizing agents to improve the tumoral response to this drug. ETO creates DNA double-strand breaks (DSB) via inhibition of DNA topoisomerase II (Top2). To repair DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ), involving D-NHEJ (dependent of the catalytic subunit of DNA-dependent protein kinase, DNA-PKcs) and B-NHEJ (backup repair pathway) are activated. We evaluated the progression of the DNA damage induced by the Top2 poison ETO in G2 phase of human HeLa cells after chemical inhibition of DNA-PKcs with NU7026. Compared to ETO treatment alone, this combined treatment resulted in a twofold higher rate of chromatid breaks and exchanges when analysis was performed in the following metaphase. Moreover, when analysis was performed in the second metaphase following treatment, increases in the percentage of micronuclei with H2AX (biomarker for DSB) foci in binucleated cells and dicentric chromosomes were seen. In post-mitotic G1 phase, a close association between unresolved DSB and meiotic recombination 11 homolog A (MRE11) signals was observed, demonstrating the contribution of MRE11 in the DSB repair by B-NHEJ. Hence, chemical inhibition of DNA-PKcs impaired both D-NHEJ and HR repair pathways, altering the maintenance of chromosomal integrity and cell proliferation. Our results suggest that the chemosensitizing effectiveness of the DNA-PKcs inhibitor and the survival rate of aberrant cells may contribute to the development of therapy-related tumors.
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Antineoplásicos Fitogênicos/toxicidade , Aberrações Cromossômicas/induzido quimicamente , Proteína Quinase Ativada por DNA/deficiência , Etoposídeo/toxicidade , Fase G2/efeitos dos fármacos , Fase G2/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico , Células HeLa , Histonas/metabolismo , Recombinação Homóloga , Humanos , Proteína Homóloga a MRE11 , Índice Mitótico , Ligação Proteica , Rad51 Recombinase/metabolismo , Troca de Cromátide Irmã/genéticaRESUMO
Introducción. La anemia de Fanconi es una enfermedad genética con herencia autosómica recesiva caracterizada por aplasia medular, predisposición a leucemia mieloide aguda, tumores sólidos y aumento en la inestabilidad cromosómica. Este síndrome puede considerarse como modelo biológico para analizar sustancias naturales con posible efecto genotóxico, difíciles de evaluar en células normales. Los objetivos de este estudio son describir y cuantificar las alteraciones cromosómicas estructurales inducidas por cinco flavonas, dos isoflavonas y una droga quimioterapéutica inhibidora de la topoisomerasa II, en cultivos de linfocitos de anemia de Fanconi a fin de determinar si existe un incremento en el número y tipo de daño cromosómico respecto al control. Materiales y métodos. Se analizaron los cromosomas de linfocitos estimulados con fitohemaglutinina M de una paciente con anemia de Fanconi. Se evaluaron 100 metafases de cada uno de los cultivos expuestos a las sustancias y control basal. Las alteraciones cromosómicas fueron documentadas con fotografías convencionales y digitales mediante analizador de imágenes. Se utilizó la prueba de c2 para determinar diferencias significativas entre el daño cromosómico entre cultivos expuestos y no expuestos con un valor de P <0,05. Resultados. Se encontraron 431 alteraciones cromosómicas en 1000 metafases analizadas; la genisteína tuvo el mayor efecto genotóxico, seguida de la genistina, fisetina, kaenferol, quercetina, baicaleína y miricetina. Las anomalías más frecuentes fueron las rupturas cromatídicas, rupturas cromosómicas, brechas (gaps) cromatídicas y cromosómicas, intercambios cuadri-radiales, cromosomas dicéntricos y rearreglos complejos. Conclusión. Los bioflavonoides genisteína, genistina y fisetina, presentes comúnmente en la dieta, aumentaron el número de alteraciones cromosómicas de manera significativa respecto al control en linfocitos de anemia de Fanconi.
Introduction. Fanconi anemia is an autosomal recessive disease characterized by a variety of congenital abnormalities, progressive bone marrow failure, increased chromosomal instability and higher risk to acute myeloid leukemia, solid tumors. This entity can be considered an appropriate biological model to analyze natural substances with possible genotoxic effect. The aims of this study were to describe and quantify structural chromosomal aberrations induced by 5 flavones,² isoflavones and a topoisomerase II chemotherapeutic inhibitor in Fanconi anemia lymphocytes in order to determine chromosomal numbers changes and/ or type of chromosomal damage. Materials and methods. Chromosomes stimulated by phytohaemagglutinin M, from Fanconi anemia lymphocytes, were analysed by conventional cytogenetic culture. For each chemical substance and controls, one hundred metaphases were evaluated. Chromosomal alterations were documented by photography and imaging analyzer. To statistical analysis was used chi square test to identify significant differences between frequencies of chromosomal damage of basal and exposed cell cultured a P value less than 0.05. Results. There were 431 chromosomal alterations in 1000 metaphases analysed; genistein was the more genotoxic bioflavonoid, followed in descendent order by genistin, fisetin, kaempferol, quercetin, baicalein and miricetin. Chromosomal aberrations observed were: chromatid breaks, chromosomal breaks, cromatid and chromosomal gaps, quadriratials exchanges, dicentrics chromosome and complex rearrangements. Conclusion. Bioflavonoids as genistein, genistin and fisetin, which are commonly present in the human diet, showed statistical significance in the number of chromosomal aberrations in Fanconi anemia lymphocytes, regarding the basal damage.