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BACKGROUND: Atherosclerosis, serving as the primary pathological mechanism at the core of cardiovascular disease, is now widely acknowledged to be associated with DNA damage and repair, contributing to atherosclerotic plaque formation. Therefore, molecules involved in the DNA repair process may play an important role in the progression of atherosclerosis. Our research endeavors to explore the contributions of specific and interrelated molecules involved in DNA repair (APE1, BRCA1, ERCC2, miR-221-3p, miR-145-5p, and miR-155-5p) to the development of atherosclerotic plaque and their interactions with each other. METHODS & RESULTS: Gene expression study was conducted using the real-time polymerase chain reaction (qRT-PCR) method on samples from carotid artery atherosclerotic plaques and nonatherosclerotic internal mammary arteries obtained from 50 patients diagnosed with coronary artery disease and carotid artery disease. Additionally, 50 healthy controls were included for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Although no difference was observed in mRNA gene expressions, we noted a decrease in miR-155-5p gene expression (p = 0.003) and an increase in miR-221-3p gene expression (p = 0.015) in plaque samples, while miR-145-5p gene expression remained unchanged (p = 0.57). Regarding serum 8-OHdG levels, patients exhibited significantly higher levels (1111.82 ± 28.64) compared to controls (636.23 ± 24.23) (p < 0.0001). CONCLUSIONS: In our study demonstrating the role of miR-155-5p and miR-221-3p in atherosclerosis, we propose that these molecules are potential biomarkers and therapeutic targets for coronary artery diseases and carotid artery disease.
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Reparación del ADN , MicroARNs , Placa Aterosclerótica , Humanos , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Femenino , Masculino , Persona de Mediana Edad , Reparación del ADN/genética , MicroARNs/genética , MicroARNs/metabolismo , Anciano , Estudios Transversales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Daño del ADN/genética , Regulación de la Expresión Génica/genética , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , 8-Hidroxi-2'-Desoxicoguanosina/metabolismoRESUMEN
APE1, an essential enzyme for DNA repair, is overexpressed in various cancers and has been identified as a potential biomarker for cancer diagnosis. However, detecting APE1 at low expression levels in the early stage of cancer presents a significant obstacle. Here, we introduced a novel localized Cas12a-based cascade amplification (LCas12a-CA) method. This method confined both the terminal deoxynucleotidyl transferase and the crRNA/Cas12a complex onto the surfaces of gold nanoparticles (AuNPs). This confinement not only boosts the stability of the multiple enzymes but also induces a substrate channeling effect. As a result, it significantly accelerates the reaction rate and enhances the sensitivity of APE1 detection. Upon the addition of APE1, the AP sites within the APE1 primer can be recognized and cleaved by APE1, exposing the 3'-OH ends. In the presence of LCas12a-CA, polyA sequences are generated at 3'-OH ends with the help of TdT and dATP. The sequences directly enter the Cas12a system, activating the trans-cleavage activity of Cas12a, thereby cutting the reporters on the surface of AuNPs and releasing fluorescence. Our platform demonstrates a detection limit (LOD) as low as 2.51 × 10-6 U/mL, which is more than 60 times lower than that of free Cas12a-CA. Furthermore, the LCas12a-CA exhibits enhanced resistance ability in extreme environments and has been proven effective for the detection of APE1 in clinical samples. Overall, this work offers a promising platform for robust biosensing in cancer diagnosis and prognosis.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa , Oro , Nanopartículas del Metal , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Nanopartículas del Metal/química , Oro/química , Endodesoxirribonucleasas/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Límite de Detección , Técnicas Biosensibles/métodos , Proteínas Asociadas a CRISPR , Proteínas Bacterianas/genéticaRESUMEN
BACKGROUND: Radiotherapy (RT) is a crucial treatment for head and neck cancer however, it causes adverse reactions to the normal tissue and organs adjacent to target tumor. The present study was carried out to investigate possible association of single nucleotide polymorphism in DNA repair genes with toxicity effects of radiotherapy on normal tissue. METHODS: Three hundred and fifty head and neck cancer patients receiving radiotherapy treatment were enrolled in this study. The adverse after effects of radiotherapy on the normal tissue in the form of skin reactions were recorded. Single nucleotide polymorphisms of APE1 (rs1130409), hOGG1 (rs1052133) and Rad51 (rs1801320, rs1801321) genes were studied by polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) and direct DNA sequencing methods and their association with development of severe radio-toxicity effects was evaluated logistic regression analysis. RESULTS: The 172G/T polymorphism of Rad51 was 2.85 times higher and significantly associated with skin reactions (OR=2.85, 95% CI: 1.50-5.41; p=0.001) and severe oral mucositis (OR=4.96, 95% CI: 2.40-10.25; p<0.0001). These results suggested that the polymorphic nature of Rad51 is responsible for risk of radiotherapy adverse effects in HNC patients. The variant 326Cys and heterozygous 326Ser/Cys genotype of hOGG1 was significantly associated with high tumor grade (OR=3.16 95% CI: 1.66-5.99; p=0.0004, and OR=3.97 95% CI: 2.15-7.34; p=<0.0001 respectively). The homozygous variant 172TT genotype of Rad51 showed positive association with poor response of both tumor and nodes towards radiotherapy treatment (p=0.007 and p=0.022). CONCLUSIONS: Interpretation of our results revealed significant association of rs1801321 SNP of Rad51 with development of adverse toxicity reactions in normal tissue of head and neck cancer patients treated with radiotherapy.
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ADN Glicosilasas , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Neoplasias de Cabeza y Cuello , Polimorfismo de Nucleótido Simple , Recombinasa Rad51 , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Masculino , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Femenino , Recombinasa Rad51/genética , Persona de Mediana Edad , ADN Glicosilasas/genética , Estudios de Seguimiento , Pronóstico , Traumatismos por Radiación/genética , Traumatismos por Radiación/etiología , Anciano , Adulto , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/patología , Genotipo , Reparación del ADN/genética , Biomarcadores de Tumor/genética , Radioterapia/efectos adversosRESUMEN
DNA walkers have emerged as a powerful tool in various biosensors, enabling the detection of low-abundance analytes with their precise programmability and efficient signal amplification capacity. However, many existing approaches are hampered by limited reaction kinetics. Herein, we designed a stochastic bipedal dual-DNA walkers (SBDW) that can traverse at high speed on AuNP-based three-dimensional (3D) tracks powered by Exo III. The SBDW exhibited superior reaction kinetics and are up to least 2.25 times faster than traditional DNA walkers, reaching a plateau within 40 min. This advancement allows for rapid and highly sensitive fluorescence detection of a significant base excision repair enzyme of APE1 with a detection limit of 0.001 U/mL. In comparison to traditional DNA walkers, this platform enables highly sensitive and specific APE1 assays in cell lysate and facilitates rapid and accurate screening of APE1 inhibitors. Given its rapid, sensitive, specific, and reliable analysis features, the strategy shows great promise in drug discovery and clinical diagnosis.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN , Inhibidores Enzimáticos , Procesos Estocásticos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , ADN/química , ADN/análisis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Técnicas Biosensibles/métodos , Oro/química , Límite de Detección , Nanopartículas del Metal/química , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , CinéticaRESUMEN
Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder associated with the amyloid beta (Aß) and tau hallmarks. The molecular insights into how neuroinflammation is initially triggered and how it affects neuronal cells are yet at the age of infancy. In this study, SH-SY5Y cells were used as a model for neurons by differentiating and were co-cultured with differentiated THP1 cells (microglia model) as well as treated with Aß(25-35) and with antioxidant FA to study inflammatory, oxidative stress responses and their effects on co-cultured neurons. Neurons co-cultured with microglial cells showed pronounced increase in ROS levels, NOS expression, truncated N-terminal form (34 kDa) of APE1 expression and AIF's translocation in the nucleus. The pre-treatment of FA, on the other hand reversed these effects. It was further evaluated how FA/Aß treatment altered microglial phenotype that in turn affected the neurons. Microglial cells showed M1 phenotype upon Aß(25-35) stress, while FA induced M2 phenotype against Aß stress, suggesting that FA alleviated Aß induced phenotype and its associated effects in the co-cultured neurons by altering the phenotype of microglial cells and induced expression of full length (37 kDa) APE1 enzyme and inhibiting AIF's nuclear translocation, thus inhibiting apoptosis. This is the first study that revealed Aß induced cleavage of APE1 enzyme in differentiated neurons, suggesting that APE1 may be the potential early target of Aß that loses its function and exacerbates AD pathology. FA activated a fully functional form of APE1 against Aß stress. The impaired function of APE1 could be the initial mechanism by which Aß induces oxidative and inflammatory responses and dietary phytochemical FA can be a potential therapeutic strategy in managing the disease by activating APE1 that not only repairs oxidative DNA base damage but also maintains mitochondrial function and alleviates neuroinflammatory responses.
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Objective: This study aimed to explore genetic variations associated with DNA repair mechanisms to enhance the management of both oral cancer (OC) and oral precancer (OPC). Methods: A cohort of 380 patients diagnosed with OC and OPC, comprising 220 males and 160 females, was analyzed. Participants were categorized based on their tobacco-chewing habits, with corresponding control groups established. Key genetic markers investigated for polymorphisms included OGG1, APE1, and XRCC1. Results: The XRCC1 Arg280H variant demonstrated significant associations with the susceptibility to both OC and OPC across various models. Further analyses, incorporating factors such as tobacco and alcohol consumption, unveiled a correlation between the XRCC1 Arg194Trp variant and an elevated risk of developing head and neck cancer. Stratified analyses also revealed an increased risk of OC or OPC based on the specific site of the cancer. Conclusion: The study underscores the importance of XRCC1 polymorphisms, particularly XRCC1 Arg280H and XRCC1 Arg194Trp, within the genetic framework of OC and OPC. Understanding these genetic associations provides valuable insights for the potential development of targeted interventions aimed at individuals predisposed to these conditions.
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The global growth of the aging population has resulted in an increased prevalence of neurodegenerative diseases, characterized by the progressive loss of central nervous system (CNS) structure and function. Given the high incidence and debilitating nature of neurodegenerative diseases, there is an urgent need to identify potential biomarkers and novel therapeutic targets thereof. Apurinic/apyrimidinic endonuclease 1 (APE1), has been implicated in several neurodegenerative diseases, as having a significant role. Abnormal APE1 expression has been observed in conditions including Alzheimer's disease, stroke, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, and epilepsy. However, whether this dysregulation is protective or harmful remains unclear. This review aims to comprehensively review the current understanding of the involvement of APE1 in neurodegenerative diseases.
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Due to the complexity of regulatory networks of disease-related biomarkers, developing simple, sensitive, and accurate methods has remained challenging for precise diagnosis. Herein, an "AND" logic gates DNA molecular machine (LGDM) was constructed, which was powered by the catalytic hairpin assembly (CHA). It was coupled with dual-emission CdTe quantum dots (QDs)-based cation exchange reaction (CER) for label-free, sensitive, and ratiometric fluorescence detection of APE1 and miRNA biomarkers. Benefiting from synergistic signal amplification strategies and a ratiometric fluorometric output mode, this LGDM enables accurate logic computing with robust and significant output signals from weak inputs. It offers improved sensitivity and selectivity even in cell extracts. Using dual-emission spectra CdTe QDs, with a ratiometric signal output mode, ensured good stability and effectively prevented false-positive signals from intrinsic biological interferences compared to the approach relying on a single signal output mode, which enabled the LGDM to achieve rapid, efficient, and accurate natural drug screening against APE1 inhibitors in vitro and cells. The developed method provides impetus to streamline research related to miRNA and APE1, offering significant promise for widespread application in drug development and clinical analysis.
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Compuestos de Cadmio , ADN-(Sitio Apurínico o Apirimidínico) Liasa , MicroARNs , Puntos Cuánticos , Telurio , Humanos , MicroARNs/análisis , MicroARNs/antagonistas & inhibidores , Telurio/química , Puntos Cuánticos/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Compuestos de Cadmio/química , Espectrometría de Fluorescencia , ADN/química , Fluorescencia , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Colorantes Fluorescentes/química , Evaluación Preclínica de Medicamentos , Computadores MolecularesRESUMEN
The resistance of osteosarcoma (OS) to ionizing radiation (IR) is an obstacle for effective patient treatment. Apurinic/apyrimidinic endonuclease-reduction/oxidation factor 1 (APE1/Ref-1) is a multifunctional protein with DNA repair and reduction/oxidation (redox) activities. We previously revealed the role of APE1 in OS radioresistance; however, whether the redox activity of APE1 is involved in OS radioresistance is unclear. APE1 regulates the activation of ataxia-telangiectasia mutated (ATM), an initiator of DNA damage response that mediates radioresistance in other cancers. The role of APE1 redox activity and ATM activation in OS radioresistance is unknown. Our study revealed that IR increased APE1 expression and ATM activation in OS cells, and APE1 directly regulated ATM activation by its redox activity. The combined use of an APE1 redox inhibitor and ATM inhibitor effectively sensitized OS cells to IR in vitro and in vivo. Mechanistically, the increased radiosensitization of OS cells by the combined use of the two inhibitors was mediated by increased ferroptosis. Co-treatment with the two inhibitors significantly decreased expression of the common targeted transcription factor P53 compared with single inhibitor treatment. Collectively, APE1 redox activity, ATM activation and their crosstalk play important roles in the resistance of OS to irradiation. Synergetic inhibition of APE1 redox activity and ATM activation sensitized OS cells to IR by inducing ferroptosis, which provides a promising strategy for OS radiotherapy.
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Proteínas de la Ataxia Telangiectasia Mutada , Neoplasias Óseas , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Ferroptosis , Osteosarcoma , Oxidación-Reducción , Radiación Ionizante , Osteosarcoma/radioterapia , Osteosarcoma/metabolismo , Osteosarcoma/tratamiento farmacológico , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Humanos , Ferroptosis/efectos de los fármacos , Línea Celular Tumoral , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/radioterapia , Tolerancia a Radiación/efectos de los fármacos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Propionatos , BenzoquinonasRESUMEN
BACKGROUND: Non-small cell lung cancer (NSCLC) represents a highly immunogenic malignancy. Immunologic tolerance facilitated by myeloid-derived suppressor cells (MDSCs) is implicated in primary or secondary resistance mechanisms in NSCLC. The potential role of APE1 in regulating NSCLC metastasis by targeting MDSCs remains uncertain. METHODS: This study utilized a plasmid, Plxpsp-mGM-CSF, to induce elevated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in A549 cells. Tumor transplantation experiments involved A549, A549+GM-CSF, and A549+GM-CSF-siAPE1 cell lines. Evaluation encompassed MDSCs, Treg cells, IgG, CD3, and CD8 levels. RESULTS: Notably, lung cancer tissues and cells displayed markedly reduced APE1 expression. siAPE1 transfection significantly curtailed tumor growth compared to the A549+GM-CSF group. APE1 knockdown orchestrated immune system modulation in lung tumor mice, characterized by diminished MDSCs but augmented Treg cells, IgG, CD3, and CD8. Additionally, APE1 knockdown led to reduced levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12) and a concurrent upregulation of the anti-MDSC cytokine IL-1ra. Furthermore, APE1 knockdown impeded cell viability in both A549 and H1650 cells. CONCLUSIONS: Transplantation of A549-GM-CSF amplified MDSC levels, fostering accelerated tumor growth, while mitigating MDSC levels through APE1 knockdown hindered tumor progression and alleviated inflammatory infiltration in lung cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach for lung cancer prevention and treatment, presenting novel insights for NSCLC management.
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Carcinoma de Pulmón de Células no Pequeñas , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Neoplasias Pulmonares , Células Supresoras de Origen Mieloide , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Animales , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/metabolismo , Humanos , Ratones , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Células A549 , Técnicas de Silenciamiento del Gen , Metástasis de la Neoplasia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Masculino , FemeninoRESUMEN
A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living cells. However, existing FA-HCR methods usually face some problems, such as a complicated design and significant background leakage, which greatly limit their application. Herein, we developed an FA-centered HCR (FAC-HCR) method based on a remote toehold-mediated strand displacement reaction. Compared to traditional HCRs mediated by four hairpin probes (HPs) and two HPs, the FAC-HCR displayed significantly decreased background leakage and improved sensitivity. Furthermore, the FAC-HCR was used to test a non-nucleic acid target, apurinic/apyrimidinic endonuclease 1 (APE1), an important BER-involved endonuclease. The fluorescence analysis results confirmed that FAC-HCR can reach a detection limit of 0.1174 U/mL. By using the two HPs for FAC-HCR with polyetherimide-based nanoparticles, the activity of APE1 in living cells can be imaged. In summary, this study could provide a new idea to design an FA-based HCR and improve the performance of HCRs in live cell imaging.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Hibridación de Ácido Nucleico , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Colorantes FluorescentesRESUMEN
Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA-APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN , Humanos , Sitios de Unión , ADN/metabolismo , ADN/química , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Microscopía de Fuerza Atómica , Unión ProteicaRESUMEN
Gastric cancer is a highly immunogenic malignancy. Immune tolerance facilitated by myeloid-derived suppressor cells (MDSCs) has been implicated in gastric cancer resistance mechanisms. The potential role of APE1 in regulating gastric cancer metastasis by targeting MDSCs remains uncertain. In this study, the plasmid Plxpsp-mGM-CSF was used to induce high expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in GES-1 cells. For tumor transplantation experiments, AGS, AGS+GM-CSF and AGS+GM-CSF-siAPE1 cell lines were established by transfection, followed by subcutaneous implantation of tumor cells. MDSCs, Treg cells, IgG, CD3 and CD8 levels were assessed. Transfection with siAPE1 significantly inhibited tumor growth compared to the AGS+GM-CSF group. APE1 gene knockdown modulated the immune system in gastric cancer mice, characterized by a decrease in MDSCs and an increase in Treg cells, IgG, CD3 and CD8. In addition, APE1 gene knockdown resulted in decreased levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12). Furthermore, APE1 gene knockdown inhibited proliferation, migration and invasion of AGS and MKN45 cells. AGS-GM-CSF cell transplantation increased MDSC levels and accelerated tumor growth, whereas APE1 knockdown reduced MDSC levels, inhibited tumor growth and attenuated inflammatory infiltration in gastric cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach to the prevention and treatment of gastric cancer, providing new insights into its management.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Células Supresoras de Origen Mieloide , Neoplasias Gástricas , Neoplasias Gástricas/patología , Neoplasias Gástricas/genética , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismo , Animales , Células Supresoras de Origen Mieloide/metabolismo , Línea Celular Tumoral , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Ratones , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Técnicas de Silenciamiento del Gen , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Metástasis de la Neoplasia , Ratones Endogámicos BALB C , Citocinas/metabolismo , Proliferación Celular/genéticaRESUMEN
APE1 is an essential gene involved in DNA damage repair, the redox regulation of transcriptional factors (TFs) and RNA processing. APE1 overexpression is common in cancers and correlates with poor patient survival. Stress granules (SGs) are phase-separated cytoplasmic assemblies that cells form in response to environmental stresses. Precise regulation of SGs is pivotal to cell survival, whereas their dysregulation is increasingly linked to diseases. Whether APE1 engages in modulating SG dynamics is worthy of investigation. In this study, we demonstrate that APE1 colocalizes with SGs and promotes their formation. Through phosphoproteome profiling, we discover that APE1 significantly alters the phosphorylation landscape of ovarian cancer cells, particularly the phosphoprofile of SG proteins. Notably, APE1 promotes the phosphorylation of Y-Box binding protein 1 (YBX1) at S174 and S176, leading to enhanced SG formation and cell survival. Moreover, expression of the phosphomutant YBX1 S174/176E mimicking hyperphosphorylation in APE1-knockdown cells recovered the impaired SG formation. These findings shed light on the functional importance of APE1 in SG regulation and highlight the importance of YBX1 phosphorylation in SG dynamics.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa , Neoplasias Ováricas , Gránulos de Estrés , Proteína 1 de Unión a la Caja Y , Femenino , Humanos , Endodesoxirribonucleasas , Neoplasias Ováricas/genética , Fosforilación , Gránulos de Estrés/metabolismo , Proteína 1 de Unión a la Caja Y/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismoRESUMEN
MicroRNA (miRNA) is involved in the progression of Alzheimer's disease (AD) and emerges as a promising AD biomarker and therapeutic target. Therefore, there is an urgent need to develop convenient and precise miRNA detection methods for AD diagnosis. Herein, a dual-signal amplification strategy based on rolling circle amplification and APE1-assisted amplification for miRNA analysis for early diagnosis of AD was proposed. The strategy consisted of dumbbell-shaped probe (DP) as amplification template and a reporter probe (RP) with an AP site modification. In the presence of the target miRNA, the miRNAs bound to the toehold domain of DP and DP was activated into a circular template. Then, RCA reaction was triggered, producing a large number of long-stranded products containing repeated sequences. After RCA, APE1 enzyme recognized and removed AP site in the complex of RCA/RP products. By coupling RCA with APE1-assisted amplification, this method has high sensitivity with the limit of detection (LOD) of 1.82 fM. Moreover, by using DP as template for RCA reaction, high specificity can be achieved. By detecting miR-206 in serum using this method, the expression of miR-206 can be accurately distinguished between AD patients and healthy individuals, indicating that this method has broad application prospects in clinical diagnosis.
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Enfermedad de Alzheimer , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/análisis , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/genética , Límite de Detección , Diagnóstico Precoz , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
In situ imaging of microRNA (miRNA) content and distribution is valuable for monitoring tumor progression. However, tumor specific in situ imaging remains a challenge due to low miRNA abundance, lack of biological compatibility, and poor specificity. In this study, we designed a DNA tetrahedral framework complex with hairpins (DTF-HPAP) consisting of an apurinic/apyrimidinic site (AP site) that could be specifically recognized and cleaved by apurinic/apyrimidinic endonuclease 1 (APE1). Efficient and specific in situ imaging of miR-21 in tumors was thus achieved through catalytic hairpin assembly (CHA) reaction. In this study, DTF-HPAP was successfully constructed to trigger the cumulative amplification of fluorescence signal in situ. The specificity, sensitivity and serum stability of DTF-HPAP were verified in vitro, and DTF-HPAP could be easily taken up by cells, acting as a biosensor to detect tumors in mice. Furthermore, we verified the ability of DTF-HPAP to specifically image miR-21 in tumors, and demonstrated its capability for tumor-specific imaging in clinical samples.
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Técnicas Biosensibles , MicroARNs , Neoplasias , Ratones , Animales , MicroARNs/genética , Endonucleasas , Catálisis , Técnicas Biosensibles/métodosRESUMEN
The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW 4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.
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Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Transporte de Proteínas , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Células HeLa , Endosomas/metabolismo , Células A549 , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Cuerpos Multivesiculares/metabolismo , Reparación por Escisión , Ácidos HidroxámicosRESUMEN
Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/REF-1) is a multifunctional protein acting on cellular signaling pathways, including DNA repair and redox activities. APE1/REF-1 has emerged as a target for cancer therapy, and its role in breast cancer models would reveal new strategies for cancer therapy. APX2009 is a specific APE1/REF-1 redox inhibitor whose anticancer properties have not been described in breast cancer cells. Here, we investigated the effect of the APX2009 treatment in the breast cancer cell lines MDA-MB-231 and MCF-7. Breast cancer cell lines were cultured, and WST1 and colony formation assays were performed to evaluate cell proliferation. Annexin V-FITC/7-AAD and LDH-Glo™ assays were performed to evaluate cell death. The wound healing assay and Matrigel transwell assay were performed after APX2009 treatment to evaluate the cellular migration and invasion processes, respectively. Our findings demonstrated that APX2009 treatment decreased breast cancer cell proliferative, migratory, and invasive properties. Furthermore, it induced apoptosis in both cell lines. Our study is the first to show the effects of APX2009 treatment on apoptosis in a breast cancer cell. Therefore, this study suggested that APX2009 treatment is a promising anticancer molecule for breast cancer.
RESUMEN
The reversible oxidation of methionine plays a crucial role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can destabilize and abrogate their function. The highly conserved methionine sulfoxide reductases protect proteins from oxidative damage by reducing their oxidized methionines, thus restoring their stability and function. Deletion or mutation in conserved methionine sulfoxide reductases leads to aging and several human neurological disorders and also reduces yeast growth on nonfermentable carbon sources. Despite their importance in human health, limited information about their physiological substrates in humans and yeast is available. For the first time, we show that Mxr2 interacts in vivo with two core proteins of the cytoplasm to vacuole targeting (Cvt) autophagy pathway, Atg19, and Ape1 in Saccharomyces cerevisiae. Deletion of MXR2 induces instability and early turnover of immature Ape1 and Atg19 proteins and reduces the leucine aminopeptidase activity of Ape1 without affecting the maturation process of Ape1. Additonally, Mxr2 interacts with the immature Ape1, dependent on Met17 present within the propeptide of Ape1 as a single substitution mutation of Met17 to Leu abolishes this interaction. Importantly, Ape1 M17L mutant protein resists oxidative stress-induced degradation in WT and mxr2Δ cells. By identifying Atg19 and Ape1 as cytosolic substrates of Mxr2, our study maps the hitherto unexplored connection between Mxr2 and the Cvt autophagy pathway and sheds light on Mxr2-dependent oxidative regulation of the Cvt pathway.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Autofagia , Metionina/metabolismo , Metionina Sulfóxido Reductasas/genética , Metionina Sulfóxido Reductasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Citoplasma/metabolismo , Vacuolas/metabolismo , Estrés Oxidativo , Estabilidad ProteicaRESUMEN
BACKGROUND: The role of DNA repair mechanisms is of significant importance in diseases characterized by elevated oxidative DNA damage, such as chronic kidney disease. It is imperative to thoroughly understand the functions of molecules associated with DNA repair mechanisms, not only for assessing susceptibility to diseases but also for monitoring disease progression. In this research, we investigated the APE1 and OGG1 gene expression levels, both of which are involved in the base excision repair (BER) mechanism in chronic hemodialysis patients with malignancy (HPM; n = 8) and without malignancy (HP; n = 36) in pre- and post-dialysis period and 37 healty persons. We also assessed how these values correlate with the clinical profiles of the patients. METHODS AND RESULTS: We conducted gene expression analysis using real-time polymerase chain reaction (qRT-PCR). No significant differences in APE1 gene expression levels were observed in pre-dialysis when comparing the HP and HPM groups to the control group. The expression levels of the OGG1 gene were significantly lower in both the HP and HPM groups in pre- and post-dialysis periods compared to the control group. Dialysis procedures led to a reduction in APE1 and OGG1 gene expression levels in both HP and HPM groups. CONCLUSIONS: The findings of our study elucidate the impact of alterations in the base excision repair (BER) mechanism, including the hemodialysis process, in end-stage renal disease (ESRD).