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1.
Sci Adv ; 10(27): eadh9613, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38959318

RESUMO

Downstream-of-gene (DoG) transcripts are an emerging class of noncoding RNAs. However, it remains largely unknown how DoG RNA production is regulated and whether alterations in DoG RNA signatures exist in major cancers. Here, through transcriptomic analyses of matched tumors and nonneoplastic tissues and cancer cell lines, we reveal a comprehensive catalog of DoG RNA signatures. Through separate lines of evidence, we support the biological importance of DoG RNAs in carcinogenesis. First, we show tissue-specific and stage-specific differential expression of DoG RNAs in tumors versus paired normal tissues with their respective host genes involved in tumor-promoting versus tumor-suppressor pathways. Second, we identify that differential DoG RNA expression is associated with poor patient survival. Third, we identify that DoG RNA induction is a consequence of treating colon cancer cells with the topoisomerase I (TOP1) poison camptothecin and following TOP1 depletion. Our results underlie the significance of DoG RNAs and TOP1-dependent regulation of DoG RNAs in diversifying and modulating the cancer transcriptome.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias , Transcriptoma , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/genética
2.
Microbes Environ ; 39(5)2024.
Artigo em Inglês | MEDLINE | ID: mdl-38839371

RESUMO

All cells must maintain the structural and functional integrity of the genome under a wide range of environments. High temperatures pose a formidable challenge to cells by denaturing the DNA double helix, causing chemical damage to DNA, and increasing the random thermal motion of chromosomes. Thermophiles, predominantly classified as bacteria or archaea, exhibit an exceptional capacity to mitigate these detrimental effects and prosper under extreme thermal conditions, with some species tolerating temperatures higher than 100°C. Their genomes are mainly characterized by the presence of reverse gyrase, a unique topoisomerase that introduces positive supercoils into DNA. This enzyme has been suggested to maintain the genome integrity of thermophiles by limiting DNA melting and mediating DNA repair. Previous studies provided significant insights into the mechanisms by which NAPs, histones, SMC superfamily proteins, and polyamines affect the 3D genomes of thermophiles across different scales. Here, I discuss current knowledge of the genome organization in thermophiles and pertinent research questions for future investigations.


Assuntos
Archaea , Bactérias , Genoma Arqueal , Genoma Bacteriano , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Bactérias/metabolismo , Genoma Bacteriano/genética , Temperatura Alta , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Reparo do DNA
3.
Nat Commun ; 15(1): 5151, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38886382

RESUMO

RNA Polymerase (RNAP) II transcription on non-coding repetitive satellite DNAs plays an important role in chromosome segregation, but a little is known about the regulation of satellite transcription. We here show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite DNAs on human centromeres. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation. Interestingly, in response to DNA double-stranded breaks (DSBs), α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner, and these DSB-induced α-satellite RNAs form into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.


Assuntos
Centrômero , DNA Topoisomerases Tipo I , DNA Satélite , RNA Polimerase II , Transcrição Gênica , Animais , DNA Satélite/genética , DNA Satélite/metabolismo , Humanos , Centrômero/metabolismo , Camundongos , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Quebras de DNA de Cadeia Dupla , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolução Molecular
4.
Biochem Biophys Res Commun ; 720: 150123, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-38759301

RESUMO

The contributions of anti-Topoisomerase 1 (Top1) autoantibodies to the pathophysiology of diffuse cutaneous systemic sclerosis (dcSSc), the most aggressive scleroderma subtype, are unknown. Top1 catalyzes DNA relaxation and unwinding in cell nuclei, a site previously considered inaccessible to antibodies. The discovery of autoantibodies in systemic lupus erythematosus that penetrate nuclei and inhibit DNA repair raised the possibility that nuclear-penetrating autoantibodies contribute to mechanisms of autoimmunity. Here we show that an anti-Top1 autoantibody produced by a single B cell clone from a patient with dcSSc penetrates live cells and localizes into nuclei. Functionally, the autoantibody inhibits formation of the Top1 cleavage complex necessary for DNA nicking, which distinguishes it from cytotoxic camptothecin Top1 inhibitors used in cancer therapy that trap the cleavage complex rather than preventing its formation. Discovery of a patient-derived cell-penetrating scleroderma anti-Top1 autoantibody that inhibits Top1 cleavage complex formation supports the hypothesis that anti-Top1 autoantibodies contribute to cellular dysfunction in dcSSc and offers a valuable antibody reagent resource for future studies on anti-Top1 autoantibody contributions to scleroderma pathophysiology.


Assuntos
Autoanticorpos , Núcleo Celular , DNA Topoisomerases Tipo I , DNA Topoisomerases Tipo I/imunologia , DNA Topoisomerases Tipo I/metabolismo , Humanos , Autoanticorpos/imunologia , Núcleo Celular/metabolismo , Esclerodermia Difusa/imunologia , Esclerodermia Difusa/tratamento farmacológico
5.
Mol Pharm ; 21(7): 3240-3255, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38785196

RESUMO

Inhibitors of a DNA repair enzyme known as polynucleotide kinase 3'-phosphatase (PNKP) are expected to show synergistic cytotoxicity in combination with topoisomerase I (TOP1) inhibitors in cancer. In this study, the synergistic cytotoxicity of a novel inhibitor of PNKP, i.e., A83B4C63, with a potent TOP1 inhibitor, i.e., SN-38, against colorectal cancer cells was investigated. Polymeric micelles (PMs) for preferred tumor delivery of A83B4C63, developed through physical encapsulation of this compound in methoxy poly(ethylene oxide)-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) micelles, were combined with SN-38 in free or PM form. The PM form of SN-38 was prepared through chemical conjugation of SN-38 to the functional end group of mPEO-b-PBCL and further assembly of mPEO-b-PBCL-SN-38 in water. Moreover, mixed micelles composed of mPEO-b-PBCL and mPEO-b-PBCL-SN-38 were used to co-load A83B4C63 and SN-38 in the same nanoformulation. The loading content (% w/w) of the SN-38 and A83B4C63 to mPEO-b-PBCL in the co-loaded formulation was 7.91 ± 0.66 and 16.13 ± 0.11% (w/w), respectively, compared to 15.67 ± 0.34 (% w/w) and 23.06 ± 0.63 (% w/w) for mPEO-b-PBCL micelles loading individual drugs. Notably, the average diameter of PMs co-encapsulating both SN-38 and A83B4C63 was larger than that of PMs encapsulating either of these compounds alone but still lower than 60 nm. The release of A83B4C63 from PMs co-encapsulating both drugs was 76.36 ± 1.41% within 24 h, which was significantly higher than that of A83B4C63-encapsulated micelles (42.70 ± 0.72%). In contrast, the release of SN-38 from PMs co-encapsulating both drugs was 44.15 ± 2.61% at 24 h, which was significantly lower than that of SN-38-conjugated PMs (74.16 ± 3.65%). Cytotoxicity evaluations by the MTS assay as analyzed by the Combenefit software suggested a clear synergy between PM/A83B4C63 (at a concentration range of 10-40 µM) and free SN-38 (at a concentration range of 0.001-1 µM). The synergistic cytotoxic concentration range for SN-38 was narrowed down to 0.1-1 or 0.01-1 µM when combined with PM/A83B4C63 at 10 or 20-40 µM, respectively. In general, PMs co-encapsulating A83B4C63 and SN-38 at drug concentrations within the synergistic range (10 µM for A83B4C63 and 0.05-1 µM for SN-38) showed slightly less enhancement of SN-38 anticancer activity than a combination of individual micelles, i.e., A83B4C63 PMs + SN-38 PMs at the same molar concentrations. This was attributed to the slower release of SN-38 from the SN-38 and A83B4C63 co-encapsulated PMs compared to PMs only encapsulating SN-38. Cotreatment of cells with TOP1 inhibitors and A83B4C63 formulation enhanced the expression level of γ-HA2X, cleaved PARP, caspase-3, and caspase-7 in most cases. This trend was more consistent and notable for PMs co-encapsulating both A83B4C63 and SN-38. The overall result from the study shows a synergy between PMs of SN-38 and A83B4C63 as a mixture of two PMs for individual drugs or PMs co-encapsulating both drugs.


Assuntos
Neoplasias Colorretais , Irinotecano , Micelas , Inibidores da Topoisomerase I , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Irinotecano/farmacologia , Irinotecano/administração & dosagem , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase I/administração & dosagem , Inibidores da Topoisomerase I/química , Linhagem Celular Tumoral , Animais , Camundongos , Nanomedicina/métodos , Sinergismo Farmacológico , DNA Topoisomerases Tipo I/metabolismo , Nanopartículas/química , Ensaios Antitumorais Modelo de Xenoenxerto , Poliésteres/química , Fosfotransferases (Aceptor do Grupo Álcool) , Enzimas Reparadoras do DNA
6.
Sci Rep ; 14(1): 10354, 2024 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710779

RESUMO

Anti-topoisomerase-I antibody (ATA) is associated with disease severity and internal organ involvement in patients with systemic sclerosis (SSc). The correlation between ATA levels and the clinical course of SSc is unclear. We aimed to determine the correlation between ATA level and survival time and the onset of internal organ fibrosis in SSc patients. This historical cohort study was conducted in adult SSc patients with quantitative tests of ATA between January 2019 and December 2022. Patients with overlap syndrome and no quantitative ATA test were excluded. According to the sample size calculation, and 10% compensated for missing data, a total of 153 patients were needed. The respective mean age on the study date and median ATA level was 59.9 ± 11.3 years and 370 U/mL (range 195-652). Most cases (107 cases; 69.9%) were the diffuse cutaneous SSc subset. According to a multivariable analysis, the ATA titer had a negative correlation with the onset of cardiac involvement (Rho - 0.47, p = 0.01), and had a positive correlation with skin thickness progression (Rho 0.39, p = 0.04). Eleven cases exhibited ATA levels < 7 U/mL and outlier ATA levels were excluded, 142 cases were included in the sensitivity analysis, and multivariable analysis showed the correlation between early onset of ILD and cardiac involvement (Rho - 0.43, p = 0.03 and Rho - 0.51, p = 0.01, respectively). The ATA level was correlated with neither the survival time nor the onset of renal crisis in both analyses. High ATA levels were correlated with a short onset of ILD and cardiac involvement and the presence of extensive skin tightness. Quantitative tests of ATA could serve as an effective tool for identifying patients at risk of an unfavorable prognosis.


Assuntos
Autoanticorpos , DNA Topoisomerases Tipo I , Escleroderma Sistêmico , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Autoanticorpos/sangue , Autoanticorpos/imunologia , DNA Topoisomerases Tipo I/imunologia , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/complicações , População do Sudeste Asiático , Tailândia/epidemiologia
7.
Chemistry ; 30(39): e202401400, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38736421

RESUMO

Coumestan represents a biologically relevant structural motif distributed in a number of natural products, and the rapid construction of related derivatives as well as the characterization of targets would accelerate lead compound discovery in medicinal chemistry. In this work, a general and scalable approach to 8,9-dihydroxycoumestans via two-electrode constant current electrolysis was developed. The application of a two-phase (aqueous/organic) system plays a crucial role for success, protecting the sensitive o-benzoquinone intermediates from over-oxidation. Based on the structurally diverse products, a primary SAR study on coumestan scaffold was completed, and compound 3 r exhibited potent antiproliferative activities and a robust topoisomerase I (Top1) inhibitory activity. Further mechanism studies demonstrates that compound 3 r was a novel Top1 poison, which might open an avenue for the development of Top1-targeted antitumor agent.


Assuntos
Antineoplásicos , Cumarínicos , DNA Topoisomerases Tipo I , Inibidores da Topoisomerase I , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase I/síntese química , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/química , Humanos , Relação Estrutura-Atividade , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Cumarínicos/química , Cumarínicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Oxirredução , Umbeliferonas/química , Umbeliferonas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais
8.
Bioorg Chem ; 147: 107412, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38696845

RESUMO

The development of novel topoisomerase I (TOP1) inhibitors is crucial for overcoming the drawbacks and limitations of current TOP1 poisons. Here, we identified two potential TOP1 inhibitors, namely, FTY720 (a sphingosine 1-phosphate antagonist) and COH29 (a ribonucleotide reductase inhibitor), through experimental screening of known active compounds. Biological experiments verified that FTY720 and COH29 were nonintercalative TOP1 catalytic inhibitors that did not induce the formation of DNA-TOP1 covalent complexes. Molecular docking revealed that FTY720 and COH29 interacted favorably with TOP1. Molecular dynamics simulations revealed that FTY720 and COH29 could affect the catalytic domain of TOP1, thus resulting in altered DNA-binding cavity size. The alanine scanning and interaction entropy identified Arg536 as a hotspot residue. In addition, the bioinformatics analysis predicted that FTY720 and COH29 could be effective in treating malignant breast tumors. Biological experiments verified their antitumor activities using MCF-7 breast cancer cells. Their combinatory effects with TOP1 poisons were also investigated. Further, FTY720 and COH29 were found to cause less DNA damage compared with TOP1 poisons. The findings provide reliable lead compounds for the development of novel TOP1 catalytic inhibitors and offer new insights into the potential clinical applications of FTY720 and COH29 in targeting TOP1.


Assuntos
Antineoplásicos , DNA Topoisomerases Tipo I , Cloridrato de Fingolimode , Simulação de Acoplamento Molecular , Inibidores da Topoisomerase I , Humanos , Cloridrato de Fingolimode/farmacologia , Cloridrato de Fingolimode/química , Cloridrato de Fingolimode/síntese química , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/química , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/síntese química , Estrutura Molecular , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Proliferação de Células/efeitos dos fármacos , Simulação de Dinâmica Molecular , Células MCF-7
9.
Cell Rep ; 43(5): 114214, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38761375

RESUMO

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Mutação , Doenças Neurodegenerativas , Diester Fosfórico Hidrolases , Humanos , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Mutação/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/genética , Transcrição Gênica , Estruturas R-Loop , Sistemas CRISPR-Cas/genética
10.
Sci Adv ; 10(21): eadm8196, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38787953

RESUMO

DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.


Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Topoisomerases Tipo I , Instabilidade Genômica , Estruturas R-Loop , RNA Polimerase II , Humanos , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Transcrição Gênica
11.
Fitoterapia ; 175: 105921, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38561052

RESUMO

Sophoridine, which is derived from the Leguminous plant Sophora alopecuroides L., has certain pharmacological activity as a new anticancer drug. Herein, a series of novel N-substituted sophoridine derivatives was designed, synthesized and evaluated with anticancer activity. Through QSAR prediction models, it was discovered that the introduction of a benzene ring as a main pharmacophore and reintroduced into a benzene in para position on the phenyl ring in the novel sophoridine derivatives improved the anticancer activity effectively. In vitro, 28 novel compounds were evaluated for anticancer activity against four human tumor cell lines (A549, CNE-2, HepG-2, and HEC-1-B). In particular, Compound 26 exhibited remarkable inhibitory effects, with an IC50 value of 15.6 µM against HepG-2 cells, surpassing cis-Dichlorodiamineplatinum (II). Molecular docking studies verified that the derivatives exhibit stronger binding affinity with DNA topoisomerase I compared to sophoridine. In addition, 26 demonstrated significant inhibition of DNA Topoisomerase I and could arrest cells in G0/G1 phase. This study provides valuable insights into the design and synthesis of N-substituted sophoridine derivatives with anticancer activity.


Assuntos
Alcaloides , Matrinas , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Quinolizinas , Sophora , Inibidores da Topoisomerase I , Humanos , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase I/síntese química , Quinolizinas/farmacologia , Quinolizinas/síntese química , Quinolizinas/química , Estrutura Molecular , Sophora/química , Alcaloides/farmacologia , Alcaloides/síntese química , Alcaloides/química , Linhagem Celular Tumoral , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/síntese química , Indolizinas/farmacologia , Indolizinas/química , Indolizinas/síntese química , DNA Topoisomerases Tipo I/metabolismo , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/síntese química
12.
Eur J Intern Med ; 125: 104-110, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38599922

RESUMO

BACKGROUND: The anti-Nucleolar Organizer Region 90 antibodies (NOR90) are rare antinuclear antibodies (ANA) reported in systemic sclerosis (SSc). Especially due to low prevalence, the clinical relevance of NOR90 in SSc remains uncertain. OBJECTIVES: To analyze the clinical associations of NOR90 in patients with SSc in a multicentric cohort. METHODS: Post-hoc, cross-sectional study of prospectively collected data from the European Scleroderma Trials and Research (EUSTAR) database, with additional information on NOR90. Further, we performed a systematic literature search, using the terms "systemic sclerosis" and "NOR90" across three databases: Medline via PubMed, Scopus, and Thomson Reuters' Web of Science Core Collection, from inception to November 1st, 2023. RESULTS: Overall, 1318 patients with SSc were included (mean age 58.3 ± 13.7 years, 81.3 % female), of whom 44 (3.3 %) were positive for NOR90. Of these, 32 were also positive for one of the SSc-criteria antibodies: 9/44 (20.5 %) for anti-topoisomerase I, 18/42 (42.9 %) for anti-centromere, and 5/40 (12.5 %) for anti-RNA polymerase III. NOR90-positive patients were more frequently female, had lower modified Rodnan skin score (mRSS), and lower prevalence of upper and lower gastrointestinal (GI) symptoms compared to NOR90-negative patients. In multivariable analysis, NOR90 remained significantly associated with lower mRSS and less frequent GI symptoms. The literature search identified 17 articles, including a total number of 87 NOR90-positive out of 3357 SSc patients, corresponding to an overall prevalence of 2.6 %. CONCLUSION: To our best knowledge, this is the largest SSc cohort tested for NOR90 to date, confirming the NOR90 prevalence in SSc patients is around 3 %.


Assuntos
Anticorpos Antinucleares , Escleroderma Sistêmico , Humanos , Escleroderma Sistêmico/imunologia , Anticorpos Antinucleares/sangue , Feminino , Pessoa de Meia-Idade , Masculino , Idoso , Estudos Transversais , Adulto , Europa (Continente) , DNA Topoisomerases Tipo I/imunologia , Relevância Clínica
13.
Front Immunol ; 15: 1339380, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38571953

RESUMO

Controlled generation of cytotoxic reactive oxygen species (ROS) is essential in cancer therapy. Ultrasound (US)-triggered sonodynamic therapy (SDT) has shown considerable ability to trigger in situ ROS generation. Unfortunately, US therapy alone is insufficient to trigger an efficient anticancer response, owing to the induction of multiple immunosuppressive factors. It was identified that 7-ethyl-10-hydroxycamptothecin (SN38) could notably inhibit DNA topoisomerase I, induce DNA damage and boost robust anticancer immunity. However, limited by the low metabolic stability, poor bioavailability, and dose-limiting toxicity, the direct usage of SN38 is inadequate in immune motivation, which limits its clinical application. Hence, new strategies are needed to improve drug delivery efficiency to enhance DNA topoisomerase I inhibition and DNA damage and elicit a vigorous anticancer cancer immunity response. Considering US irradiation can efficiently generate large amounts of ROS under low-intensity irradiation, in this study, we aimed to design a polymeric, ROS-responsive SN38 nanoformulation for in vivo drug delivery. Upon the in-situ generation of ROS by US therapy, controlled on-demand release of SN38 occurred in tumor sites, which enhanced DNA damage, induced DC cell maturation, and boosted anticancer immunity. Our results demonstrated that a new strategy of involving the combination of a SN38 nanoformulation and US therapy could be used for cancer immunotherapy.


Assuntos
Nanopartículas , Neoplasias , Espécies Reativas de Oxigênio/metabolismo , DNA Topoisomerases Tipo I , Linhagem Celular Tumoral , Imunoterapia , Neoplasias/terapia
14.
J Med Chem ; 67(9): 7006-7032, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38668707

RESUMO

G-quadruplexes are noncanonical four-stranded DNA secondary structures. MYC is a master oncogene and the G-quadruplex formed in the MYC promoter functions as a transcriptional silencer and can be stabilized by small molecules. We have previously revealed a novel mechanism of action for indenoisoquinoline anticancer drugs, dual-downregulation of MYC and inhibition of topoisomerase I. Herein, we report the design and synthesis of novel 7-aza-8,9-methylenedioxyindenoisoquinolines based on desirable substituents and π-π stacking interactions. These compounds stabilize the MYC promoter G-quadruplex, significantly lower MYC levels in cancer cells, and inhibit topoisomerase I. MYC targeting was demonstrated by differential activities in Raji vs CA-46 cells and cytotoxicity in MYC-dependent cell lines. Cytotoxicities in the NCI-60 panel of human cancer cell lines were investigated. Favorable pharmacokinetics were established, and in vivo anticancer activities were demonstrated in xenograft mouse models. Furthermore, favorable brain penetration, brain pharmacokinetics, and anticancer activity in an orthotopic glioblastoma mouse model were demonstrated.


Assuntos
Antineoplásicos , Desenho de Fármacos , Quadruplex G , Isoquinolinas , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc , Inibidores da Topoisomerase I , Quadruplex G/efeitos dos fármacos , Humanos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Isoquinolinas/farmacologia , Isoquinolinas/química , Isoquinolinas/farmacocinética , Isoquinolinas/síntese química , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/farmacocinética , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/uso terapêutico , Relação Estrutura-Atividade , DNA Topoisomerases Tipo I/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
DNA Repair (Amst) ; 137: 103664, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38484460

RESUMO

The type IB topoisomerase of budding yeast (yTop1) generates small deletions in tandem repeats through a sequential cleavage mechanism and larger deletions with random endpoints through the nonhomologous end-joining (NHEJ) pathway. Vaccinia virus Top1 (vTop1) is a minimized version of the eukaryal TopIB enzymes and uniquely has a strong consensus cleavage sequence: the pentanucleotide (T/C)CCTTp↓. To define the relationship between the position of TopIB cleavage and mutagenic outcomes, we expressed vTop1 in yeast top1Δ strains containing reporter constructs with a single CCCTT site, tandem CCCTT sites, or CCCTT sites separated by 42 bp. vTop1 cleavage at a single CCCTT site was associated with small, NHEJ-dependent deletions. As observed with yTop1, vTop1 generated 5-bp deletions at tandem CCCTT sites. In contrast to yTop1-initiated deletions, however, 5-bp deletions associated with vTop1 expression were not affected by the level of ribonucleotides in genomic DNA. vTop1 expression was associated with a 47-bp deletion when CCCTT sites were separated by 42 bp. Unlike yTop1-initiated large deletions, the vTop1-mediated 47-bp deletion did not require NHEJ, consistent with a model in which re-ligation of enzyme-associated double-strand breaks is catalyzed by vTop1.


Assuntos
Saccharomyces cerevisiae , Vaccinia virus , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vaccinia virus/genética , Vaccinia virus/metabolismo , DNA/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Mutagênese , Proteínas Virais/metabolismo
16.
Clin Immunol ; 262: 110195, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38494058

RESUMO

CD21low B cells have recently been found increased in SSc-associated digital ulcers (DUs) or interstitial lung disease (ILD). To further characterize CD21low B cells which encompass autoreactive cells, we analyzed their expression of the inhibitory CD32 receptor in SSc. Peripheral blood mononuclear cells from 27 patients with SSc and 15 age-and sex-matched healthy controls (HCs) were analyzed with multicolor flow cytometry. CD21low B cells were significantly increased in patients with DUs (51.3%) compared to HCs (28.1%) and in patients with ILD (53.1%) compared to HCs. CD21lowCD32low B cells were significantly increased in patients with DUs (23.8%) compared to HCs (4.4%), in patients with ILD (28.4%) compared to HCs, and in anti-topoisomerase I (+) patients (21.5%) compared to HCs and to anti-topoisomerase I (-) patients (2.4%). Autoreactive B cells recognizing Topoisomerase I were predominantly within CD32low cell fraction. Our study further supports the autoreactive status of CD21lowCD32low B cells in SSc patients.


Assuntos
DNA Topoisomerases Tipo I , Doenças Pulmonares Intersticiais , Proteínas Nucleares , Escleroderma Sistêmico , Úlcera Cutânea , Humanos , Leucócitos Mononucleares
17.
Cells ; 13(6)2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38534397

RESUMO

Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.


Assuntos
DNA Topoisomerases Tipo I , DNA , Humanos , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , DNA de Cadeia Simples , DNA Helicases/genética , RNA
18.
Bioorg Med Chem Lett ; 104: 129710, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38518997

RESUMO

A novel series of benzo[6,7]indolo[3,4-c]isoquinolines 3a-3f was designed by scaffold hopping of topoisomerase I inhibitor benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-ones (BBPIs), which were developed by structural modification of the natural marine product lamellarin. The unconventional pentacycle was constructed by Bischler-Napieralski-type condensation of amide 11 and subsequent intramolecular Heck reaction. In vitro anticancer activity of the synthesized benzo[6,7]indolo[3,4-c]isoquinolines was evaluated on a panel of 39 human cancer cell lines (JFCR39). Among the compounds tested, N-(3-morpholinopropyl) derivative 3e showed the most potent antiproliferative activity, with a mean GI50 value of 39 nM. This compound inhibited topoisomerase I activity by stabilizing the enzyme-DNA complex.


Assuntos
Antineoplásicos , Cumarínicos , Compostos Heterocíclicos de 4 ou mais Anéis , Isoquinolinas , Inibidores da Topoisomerase I , Humanos , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Isoquinolinas/síntese química , Isoquinolinas/química , Isoquinolinas/farmacologia , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia , Desenho de Fármacos , Cumarínicos/síntese química , Cumarínicos/química , Cumarínicos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia
19.
J Biol Chem ; 300(4): 107162, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38484800

RESUMO

Kinetoplastid parasites are "living bridges" in the evolution from prokaryotes to higher eukaryotes. The near-intronless genome of the kinetoplastid Leishmania exhibits polycistronic transcription which can facilitate R-loop formation. Therefore, to prevent such DNA-RNA hybrids, Leishmania has retained prokaryotic-like DNA Topoisomerase IA (LdTOPIA) in the course of evolution. LdTOPIA is an essential enzyme that is expressed ubiquitously and is adapted for the compartmentalized eukaryotic form in harboring functional bipartite nuclear localization signals. Although exhibiting greater homology to mycobacterial TOPIA, LdTOPIA could functionally complement the growth lethality of Escherichia coli TOPIA null GyrB ts strain at non-permissive temperatures. Purified LdTOPIA exhibits Mg2+-dependent relaxation of only negatively supercoiled DNA and preference towards single-stranded DNA substrates. LdTOPIA prevents nuclear R-loops as conditional LdTOPIA downregulated parasites exhibit R-loop formation and thereby parasite killing. The clinically used tricyclic antidepressant, norclomipramine could specifically inhibit LdTOPIA and lead to R-loop formation and parasite elimination. This comprehensive study therefore paves an avenue for drug repurposing against Leishmania.


Assuntos
DNA Topoisomerases Tipo I , Leishmania , Proteínas de Protozoários , Estruturas R-Loop , Animais , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Leishmania/enzimologia , Leishmania/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Tripanossomicidas/química , Tripanossomicidas/farmacologia
20.
Nucleic Acids Res ; 52(5): 2142-2156, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38340342

RESUMO

Human DNA topoisomerase 1 (Top1) is a crucial enzyme responsible for alleviating torsional stress on DNA during transcription and replication, thereby maintaining genome stability. Previous researches had found that non-working Top1 interacted extensively with chromosomal DNA in human cells. However, the reason for its retention on chromosomal DNA remained unclear. In this study, we discovered a close association between Top1 and chromosomal DNA, specifically linked to the presence of G-quadruplex (G4) structures. G4 structures, formed during transcription, trap Top1 and hinder its ability to relax neighboring DNAs. Disruption of the Top1-G4 interaction using G4 ligand relieved the inhibitory effect of G4 on Top1 activity, resulting in a further reduction of R-loop levels in cells. Additionally, the activation of Top1 through the use of a G4 ligand enhanced the toxicity of Top1 inhibitors towards cancer cells. Our study uncovers a negative regulation mechanism of human Top1 and highlights a novel pathway for activating Top1.


Assuntos
DNA Topoisomerases Tipo I , Quadruplex G , Transcrição Gênica , Humanos , DNA/química , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Ligantes , Inibidores da Topoisomerase I/farmacologia
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