RESUMEN
Designing and developing inhibitors against the epigenetic target DNA methyltransferase (DNMT) is an attractive strategy in epigenetic drug discovery. DNMT1 is one of the epigenetic enzymes with significant clinical relevance. Structure-based de novo design is a drug discovery strategy that was used in combination with similarity searching to identify a novel DNMT inhibitor with a novel chemical scaffold and warrants further exploration. This study aimed to continue exploring the potential of de novo design to build epigenetic-focused libraries targeted toward DNMT1. Herein, we report the results of an in-depth and critical comparison of ligand- and structure-based de novo design of screening libraries focused on DNMT1. The newly designed chemical libraries focused on DNMT1 are freely available on GitHub.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1 , Diseño de Fármacos , Inhibidores Enzimáticos , Ligandos , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
DNA methyltransferase 1 (DNMT1) is the primary enzyme responsible for maintaining DNA methylation patterns during cellular division, crucial for cancer development by suppressing tumor suppressor genes. In this study, we retained the phthalimide structure of N-phthaloyl-l-tryptophan (RG108) and substituted its indole ring with nitrogen-containing aromatic rings of varying sizes. We synthesized 3-(9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acids and confirmed them as DNMT1 inhibitors through protein affinity testing, radiometric method using tritium labeled SAM, and MTT assay. Preliminary structure-activity relationship analysis revealed that introducing substituents on the carbazole ring could enhance inhibitory activity, with S-configuration isomers showing greater activity than R-configuration ones. Notably, S-3-(3,6-di-tert-butyl-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7r-S) and S-3-(1,3,6-trichloro-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7t-S) exhibited significant DNMT1 enzyme inhibition activity, with IC50 values of 8.147 µM and 0.777 µM, respectively (compared to RG108 with an IC50 above 250 µM). Moreover, they demonstrated potential anti-proliferative activity on various tumor cell lines including A2780, HeLa, K562, and SiHa. Transcriptome analysis and KEGG pathway enrichment of K562 cells treated with 7r-S and 7t-S identified differentially expressed genes (DEGs) related to apoptosis and cell cycle pathways. Flow cytometry assays further indicated that 7r-S and 7t-S induce apoptosis in K562 cells and arrest them in the G0/G1 phase in a concentration-dependent manner. Molecular docking revealed that 7t-S may bind to the methyl donor S-adenosyl-l-methionine (SAM) site in DNMT1 with an orientation opposite to RG108, suggesting potential for deeper penetration into the DNMT1 pocket and laying the groundwork for further modifications.
Asunto(s)
Carbazoles , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1 , Inhibidores Enzimáticos , Humanos , Relación Estructura-Actividad , Carbazoles/farmacología , Carbazoles/química , Carbazoles/síntesis química , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Proliferación Celular/efectos de los fármacos , Estructura Molecular , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Ensayos de Selección de Medicamentos Antitumorales , Relación Dosis-Respuesta a Droga , Indoles/farmacología , Indoles/química , Indoles/síntesis química , Simulación del Acoplamiento Molecular , Línea Celular Tumoral , Ftalimidas , Triptófano/análogos & derivadosRESUMEN
Psoriasis is classified as an autoimmune disorder characterized by abnormal immune response leading to the development of chronic dermal inflammation. Most individuals have a genetic vulnerability that may be further influenced by epigenetic changes occurring due to multiple variables such as pollutant exposure. Epigenetic modifications such as DNA methylation possess a dynamic nature, enabling cellular differentiation and adaptation by controlling gene expression. Di(2-ethylhexyl) phthalate (DEHP) and psoriatic inflammation are known to cause modification of DNA methylation via DNA methyltransferase (DNMT). However, it is not known whether DEHP, a ubiquitous plasticizer affects psoriatic inflammation via DNMT modulation. Therefore, this study investigated the effect of DNMT inhibitor, 5-aza-2'-deoxycytidine (AZA) on DEHP-induced changes in the expression of DNMT1, global DNA methylation, and anti-/inflammatory parameters (p-STAT3, IL-17A, IL-6, iNOS, IL-10, Foxp3, Nrf2, HO-1) in the skin and the peripheral adaptive/ myeloid immune cells (CD4+ T cells/CD11b+ cells) in imiquimod (IMQ) model of psoriasiform inflammation. Further, psoriasis-associated clinical/histopathological features (ear thickness, ear weight, ear PASI score, MPO activity, and H&E staining of the ear and the back skin) were also analyzed in IMQ model. Our data show that IMQ-treated mice with DEHP exposure had increased DNMT1 expression and DNA methylation which was associated with elevated inflammatory (p-STAT3, IL-17A, IL-6, iNOS) and downregulated anti-inflammatory mediators (IL-10, Foxp3, Nrf2, HO-1) in the peripheral immune cells (CD4+ T cells/CD11b+ cells) and the skin as compared to IMQ-treated mice. Treatment with DNMT1 inhibitor caused reduction in inflammatory and elevation in anti-inflammatory parameters with significant improvement in clinical/histopathological symptoms in both IMQ-treated and DEHP-exposed IMQ-treated mice. In conclusion, our study shows strong evidence indicating that DNMT1 plays an important role in DEHP-induced exacerbation of psoriasiform inflammation in mice through hypermethylation of DNA.
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ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Decitabina , Dietilhexil Ftalato , Psoriasis , Piel , Animales , Metilación de ADN/efectos de los fármacos , Psoriasis/inducido químicamente , Psoriasis/tratamiento farmacológico , Psoriasis/inmunología , Psoriasis/patología , Decitabina/farmacología , Decitabina/uso terapéutico , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Piel/patología , Piel/efectos de los fármacos , Piel/inmunología , Dietilhexil Ftalato/toxicidad , Ratones , Masculino , Antiinflamatorios/uso terapéutico , Antiinflamatorios/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos BALB C , FemeninoRESUMEN
Bioorthogonal chemistry represents a powerful tool in chemical biology, which shows great potential in epigenetic modulation. As a proof of concept, the epigenetic modulation model of mitochondrial DNA (mtDNA) is selected because mtDNA establishes a relative hypermethylation stage under oxidative stress, which impairs the mitochondrion-based therapeutic effect during cancer therapy. Herein, we design a new biocompatible hydrogen-bonded organic framework (HOF) for a HOF-based mitochondrion-targeting bioorthogonal platform TPP@P@PHOF-2. PHOF-2 can activate a prodrug (pro-procainamide) in situ, which can specifically inhibit DNA methyltransferase 1 (DNMT1) activity and remodel the epigenetic modification of mtDNA, making it more susceptible to ROS damage. In addition, PHOF-2 can also catalyze artemisinin to produce large amounts of ROS, effectively damaging mtDNA and achieving better chemodynamic therapy demonstrated by both in vitro and in vivo studies. This work provides new insights into developing advanced bioorthogonal therapy and expands the applications of HOF and bioorthogonal catalysis.
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ADN Mitocondrial , Epigénesis Genética , Mitocondrias , Especies Reactivas de Oxígeno , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Humanos , ADN Mitocondrial/genética , Epigénesis Genética/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Enlace de Hidrógeno , Animales , Ratones , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/genética , Profármacos/farmacología , Profármacos/química , Estructuras Metalorgánicas/química , Estructuras Metalorgánicas/farmacologíaRESUMEN
DNA methylation is an epigenetic mechanism that introduces a methyl group at the C5 position of cytosine. This reaction is catalyzed by DNA methyltransferases (DNMTs) and is essential for the regulation of gene transcription. The DNMT1 and DNMT3A or -3B family proteins are known targets for the inhibition of DNA hypermethylation in cancer cells. A selective non-nucleoside DNMT3A inhibitor was developed that mimics S-adenosyl-l-methionine and deoxycytidine; however, the mechanism of selectivity is unclear because the inhibitor-protein complex structure determination is absent. Therefore, we performed docking and molecular dynamics simulations to predict the structure of the complex formed by the association between DNMT3A and the selective inhibitor. Our simulations, binding free energy decomposition analysis, structural isoform comparison, and residue scanning showed that Arg688 of DNMT3A is involved in the interaction with this inhibitor, as evidenced by its significant contribution to the binding free energy. The presence of Asn1192 at the corresponding residues in DNMT1 results in a loss of affinity for the inhibitor, suggesting that the interactions mediated by Arg688 in DNMT3A are essential for selectivity. Our findings can be applied in the design of DNMT-selective inhibitors and methylation-specific drug optimization procedures.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas , ADN Metiltransferasa 3A , Inhibidores Enzimáticos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Metilación de ADN , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/química , Sitios de UniónRESUMEN
BACKGROUND: The faithful maintenance of DNA methylation homeostasis indispensably requires DNA methyltransferase 1 (DNMT1) in cancer progression. We previously identified DNMT1 as a potential candidate target for oral squamous cell carcinoma (OSCC). However, how the DNMT1- associated global DNA methylation is exploited to regulate OSCC remains unclear. METHODS: The shRNA-specific DNMT1 knockdown was employed to target DNMT1 on oral cancer cells in vitro, as was the use of DNMT1 inhibitors. A xenografted OSCC mouse model was established to determine the effect on tumor suppression. High-throughput microarrays of DNA methylation, bulk and single-cell RNA sequencing analysis, multiplex immunohistochemistry, functional sphere formation and protein immunoblotting were utilized to explore the molecular mechanism involved. Analysis of human samples revealed associations between DNMT1 expression, global DNA methylation and collaborative molecular signaling with oral malignant transformation. RESULTS: We investigated DNMT1 expression boosted steadily during oral malignant transformation in human samples, and its inhibition considerably minimized the tumorigenicity in vitro and in a xenografted OSCC model. DNMT1 overexpression was accompanied by the accumulation of cancer-specific DNA hypomethylation during oral carcinogenesis; conversely, DNMT1 knockdown caused atypically extensive genome-wide DNA hypomethylation in cancer cells and xenografted tumors. This novel DNMT1-remodeled DNA hypomethylation pattern hampered the dual activation of PI3K-AKT and CDK2-Rb and inactivated GSK3ß collaboratively. When treating OSCC mice, targeting DNMT1 achieved greater anticancer efficacy than the PI3K inhibitor, and reduced the toxicity of blood glucose changes caused by the PI3K inhibitor or combination of PI3K and CDK inhibitors as well as adverse insulin feedback. CONCLUSIONS: Targeting DNMT1 remodels a novel global DNA hypomethylation pattern to facilitate anticancer efficacy and minimize potential toxic effects via balanced signaling synergia. Our study suggests DNMT1 is a crucial gatekeeper regarding OSCC destiny and treatment outcome.
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ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Boca , Humanos , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Animales , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Ratones , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Transducción de Señal , Proliferación CelularRESUMEN
BACKGROUND: As the variable clinical outcome of patients with hepatoblastoma (HB) cannot be explained by genetics alone, the identification of drugs with the potential to effectively reverse epigenetic alterations is a promising approach to overcome poor therapy response. The gene ubiquitin like with PHD and ring finger domains 1 (UHRF1) represents an encouraging epigenetic target due to its regulatory function in both DNA methylation and histone modifications and its clinical relevance in HB. METHODS: Patient-derived xenograft in vitro and in vivo models were used to study drug response. The mechanistic basis of CM-272 treatment was elucidated using RNA sequencing and western blot experiments. RESULTS: We validated in comprehensive data sets that UHRF1 is highly expressed in HB and associated with poor outcomes. The simultaneous pharmacological targeting of UHRF1-dependent DNA methylation and histone H3 methylation by the dual inhibitor CM-272 identified a selective impact on HB patient-derived xenograft cell viability while leaving healthy fibroblasts unaffected. RNA sequencing revealed downregulation of the IGF2-activated survival pathway as the main mode of action of CM-272 treatment, subsequently leading to loss of proliferation, hindered colony formation capability, reduced spheroid growth, decreased migration potential, and ultimately, induction of apoptosis in HB cells. Importantly, drug response depended on the level of IGF2 expression, and combination assays showed a strong synergistic effect of CM-272 with cisplatin. Preclinical testing of CM-272 in a transplanted patient-derived xenograft model proved its efficacy but also uncovered side effects presumably caused by its strong antitumor effect in IGF2-driven tumors. CONCLUSIONS: The inhibition of UHRF1-associated epigenetic traces, such as IGF2-mediated survival, is an attractive approach to treat high-risk HB, especially when combined with the standard-of-care therapeutic cisplatin.
Asunto(s)
Hepatoblastoma , Neoplasias Hepáticas , Humanos , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Cisplatino/farmacología , Hepatoblastoma/tratamiento farmacológico , Hepatoblastoma/genética , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Ubiquitina-Proteína Ligasas/genética , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidoresRESUMEN
Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1 , Conformación de Ácido Nucleico , ARN , Humanos , Cromatina/metabolismo , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ARN/genética , ARN/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/metabolismoRESUMEN
Objective: DNA methylation played a vital role in the progression of diabetic retinopathy. In this study, we aimed to explore the role of DNA cytosine-5-methyltransferase 1 (DNMT1) in the development of early diabetic retinopathy and its potential underlying mechanism. Methods: Eight-week-old healthy Mongolian gerbils were used to establish type 1 diabetes using streptozotocin (STZ). Alteration of weight, fasting blood glucose, density of RGCs (Tuj1-labeled), and H&E-stained retinal cross sections were applied to evaluate the diabetic retinopathy mouse model. The global DNA methylation level of the retina at different time points after STZ injection was measured using the global methylation assay. Western blot was used to detect the protein expression of DNMT1, DNA methyltransferase 3A (DNMT3A), and 3B (DNMT3B). Quantitative reverse transcription-polymerase chain reactions (qRT-PCR) and western blot were used to determine the expression of CDKN2B. Cell proliferation and cell cycle were evaluated by the MTS assay and flow cytometry. Results: STZ injection caused the increased global DNA methylation level, which reached a maximum at 6 weeks after injection. Moreover, STZ injection caused the damage of RGCs. At 6 weeks after STZ injection, the expression levels of DNMT1 and DNMT3B were significantly increased in the STZ group. DNMT1-induced DNA hypermethylation inhibited the expression of CDKN2B (a negative regulator of cell cycle). DNMT1-mediated DNA methylation facilitated RGC proliferation via regulating the expression of CDKN2B. Conclusion: DNMT1-mediated DNA methylation played an important role in STZ-induced diabetic retinopathy via modulating CDKN2B expression.
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Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Retinopatía Diabética/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Proliferación Celular , Biología Computacional , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/etiología , Retinopatía Diabética/patología , Técnicas de Silenciamiento del Gen , Gerbillinae , Masculino , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Estreptozocina/toxicidadRESUMEN
Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.
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Neoplasias de la Mama/patología , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Decitabina/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/análogos & derivados , Transición Epitelial-Mesenquimal , Femenino , Genes erbB-2/genética , Humanos , Concentración 50 Inhibidora , Ratones , Ratones SCID , Pruebas de Mutagenicidad , Polietilenglicoles/farmacología , Distribución Aleatoria , Trastuzumab/farmacología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ABL1 tyrosine kinase inhibitors (TKIs) dramatically improve the prognosis of chronic myeloid leukemia (CML), but 10-20% of patients achieve suboptimal responses with low TKIs sensitivity. Furthermore, residual leukemic stem cells (LSCs) are involved in the molecular relapse after TKIs discontinuation. Aberrant DNA hypermethylation contributes to low TKIs sensitivity and the persistence of LSCs in CML. DNMT1 is a key regulator of hematopoietic stem cells, suggesting that aberrant DNA hypermethylation targeting DNMT1 represents a potential therapeutic target for CML. We investigated the efficacy of OR-2100 (OR21), the first orally available single-compound prodrug of decitabine. OR21 exhibited anti-tumor effects as a monotherapy, and in combination therapy it increased TKI-induced apoptosis and induction of tumor suppressor genes including PTPN6 encoding SHP-1 in CML cells. OR21 in combination with imatinib significantly suppressed tumor growth in a xenotransplant model. OR21 and combination therapy decreased the abundance of LSCs and inhibited engraftment in a BCR-ABL1-transduced mouse model. These results demonstrate that targeting DNMT1 using OR21 exerts anti-tumor effects and impairs LSCs in CML. Therefore, combination treatment of TKIs and OR21 represents a promising treatment strategy in CML.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Animales , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Humanos , Células Jurkat , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Ratones , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Exposure to benzene or its metabolite hydroquinone (HQ) is a risk factor for a series of myeloid malignancies, and long noncoding RNAs play an important role in the process of pathogenesis. Urothelial cancer-associated 1 (UCA1) functions as an oncogene in the development of acute myeloid leukemia. However, the association between DNMT1 and UCA1 with benzene or HQ exposure has not been explored. We characterized UCA1 expression in cells briefly exposed to HQ (HQ-ST cells) and HQ-induced malignantly transformed (TK6-HT cells) treated with 5-aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA). Compared to that in control cells, UCA1 expression was increased, whereas DNMT1 was decreased in HQ-ST cells and TK6-HT cells treated with 5-AzaC or TSA. Moreover, UCA1 expression was also upregulated and positively correlated with benzene exposure time in benzene-exposed workers. Furthermore, the expression of UCA1 was negatively associated with the DNA methylation level of its promoter in benzene-exposed workers. DNMT1 rather than DNMT3b knockout in TK6-HT cells activated the expression of UCA1 by inducing its promoter hypomethylation. These results suggest that benzene or HQ exposure leads to UCA1 upregulation via DNA hypomethylation in the UCA1 promoter, which is mediated by DNMT1.
Asunto(s)
Benceno/toxicidad , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Hidroquinonas/toxicidad , Exposición Profesional , ARN Largo no Codificante/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Azacitidina/farmacología , Línea Celular , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Humanos , Ácidos Hidroxámicos/farmacología , Regiones Promotoras Genéticas , ARN Largo no Codificante/genéticaRESUMEN
Breast cancer (BC) is the most frequently diagnosed female cancer and second leading cause of death. Despite the discovery of many antineoplastic drugs for BC, the current therapy is not totally efficient. In this study, we investigated the potential of repurposing the well-known diabetes type II drug liraglutide to modulate epigenetic modifications in BC cells lines in vitro and in vivo via Ehrlich mice tumors models. The in vitro results revealed a significant reduction on cell viability, migration, DNMT activity and displayed lower levels of global DNA methylation in BC cell lines after liraglutide treatment. The interaction between liraglutide and the DNMT enzymes resulted in a decrease profile of DNA methylation for the CDH1, ESR1 and ADAM33 gene promoter regions and, consequently, increased their gene and protein expression levels. To elucidate the possible interaction between liraglutide and the DNMT1 protein, we performed an in silico study that indicates liraglutide binding in the catalytic cleft via hydrogen bonds and salt bridges with the interdomain contacts and disturbs the overall enzyme conformation. The in vivo study was also able to reveal that liraglutide and the combined treatment of liraglutide and paclitaxel or methotrexate were effective in reducing tumor growth. Moreover, the modulation of CDH1 and ADAM33 mouse gene expression by DNA demethylation suggests a role for liraglutide in DNMT activity in vivo. Altogether, these results indicate that liraglutide may be further analysed as a new adjuvant treatment for BC.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Liraglutida/uso terapéutico , Proteínas ADAM/genética , Animales , Antígenos CD/genética , Neoplasias de la Mama/patología , Cadherinas/genética , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Ratones , Regiones Promotoras GenéticasRESUMEN
DNA methyltransferase (DNMT) 1 is an enzyme that functions as a maintenance methyltransferase during DNA replication, and depletion of this enzyme from cells is considered to be a rational goal in DNA methylation-dependent disorders. Two DNMT1-depleting agents 5-aza-2'-deoxycytidine (aza-dCyd, decitabine) and 5-aza-cytidine (aza-Cyd, azacitidine) are currently used for the treatment of myelodysplastic syndromes and acute myeloid leukemia and have also been investigated for nononcology indications, such as sickle cell disease. However, these agents have several off-target activities leading to significant toxicities that limit dosing and duration of treatment. Development of more selective inhibitors of DNMT1 could therefore afford treatment of long durations at effective doses. We have discovered that 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) is as effective as aza-dCyd in depleting DNMT1 in mouse tumor models but with markedly low toxicity. In this review we describe the preclinical studies that led to the development of aza-T-dCyd as a superior DNMT1-depleting agent with respect to aza-dCyd and will describe its pharmacology, metabolism, and mechanism of action. In an effort to understand why aza-T-dCyd is a more selective DNMT1 depleting agent than aza-dCyd, we will also compare and contrast the activities of these two agents. SIGNIFICANCE STATEMENT: Aza-T-dCyd is a potent DNMT1-depleting agent. Although similar in structure to decitabine (aza-dCyd), its metabolism and mechanism of action is different than that of aza-dCyd, resulting in less off-target activity and less toxicity. The larger therapeutic index of aza-T-dCyd (DNMT1 depletion vs. toxicity) in mice suggests that it would be a better clinical candidate to selectively deplete DNMT1 from target cells and determine whether or not depletion of DNMT1 is an effective target for various diseases.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Desoxicitidina/síntesis química , Desoxicitidina/farmacología , Desarrollo de Medicamentos/métodos , Administración Oral , Animales , Disponibilidad Biológica , Desarrollo de Medicamentos/tendencias , HumanosRESUMEN
Advanced osteosarcoma (OSA) is highly aggressive and can lead to distant metastasis or recurrence. Here, a novel small-molecule inhibitor/antagonist of DNA methyltransferase 1 (DNMT-1) named DI-1 (inhibitor of DNMT-1) was explored to enhance the antitumor effect of a molecular-targeted agent, cabozantinib, on OSA cell lines. In patients with OSA, expression of DNMT-1 was negatively related with that of microRNA (miR)-34a and associated with a poor prognosis. In OSA cell lines (OSA cell line U2OS and an OSA cell line U2OSR resistance to cabozantinib), DI-1 treatment enhanced miR-34a expression by inhibiting hypermethylation of the promoter region of miR-34a mediated by DNMT-1. DI-1 enhanced the sensitivity of OSA cells (U2OS, 143B and MG63) to cabozantinib and other molecular-targeted agents by enhancing miR-34a expression and repressing activation of the Notch pathway. Mechanistically, DI-1 repressed recruitment of DNMT-1 to the promoter region of miR-34a and, in turn, decreased the methylation rate in the promoter region of miR-34a in OSA cells. These results suggest that repressing DNMT-1 activation by DI-1 enhances miR-34a expression in OSA cells and could be a promising therapeutic strategy for OSA.
Asunto(s)
Anilidas/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias Óseas/tratamiento farmacológico , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Osteosarcoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/mortalidad , Neoplasias Óseas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADN , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Ratones Desnudos , MicroARNs , Osteosarcoma/genética , Osteosarcoma/mortalidad , Osteosarcoma/patología , Pronóstico , Regiones Promotoras Genéticas , Índice de Severidad de la EnfermedadRESUMEN
The peripheral T-cell lymphomas (PTCL) could be considered the prototypical epigenetic disease. As a disease, they are uniquely sensitive to histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors, both alone and in combination, are characterized by a host of mutations in epigenetic genes, and can develop spontaneously in genetically engineered murine models predicated on established recurring mutations in (RHOAG17V) and TET2, an epigenetic gene governing DNA methylation. Given the clinical benefit of HDAC inhibitors (HDACi) and hypomethlyation agents alone and in combination in PTCL, we sought to explore a mechanistic basis for these agents in PTCL. Herein, we reveal profound class synergy between HDAC and DNMT inhibitors in PTCL, and that the combination induces degrees of gene expression that are substantially different and more extensive than that observed for the single agents. A prominent signature of the combination relates to the transcriptional induction of cancer testis antigens and genes involved in the immune response. Interestingly, TBX21 and STAT4, master regulators of TH1 differentiation, were among the genes upregulated by the combination, suggesting the induction of a TH1-like phenotype. Moreover, suppression of genes involved in cholesterol metabolism and the matrisome were also identified. We believe that these data provide a strong rationale for clinical studies, and future combinations leveraging an immunoepigenetic platform.
Asunto(s)
Antígenos de Neoplasias/genética , Biomarcadores de Tumor/metabolismo , Epigenoma , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inmunidad/genética , Linfoma de Células T/patología , Testículo/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Azacitidina/farmacología , Biomarcadores de Tumor/genética , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Metilación de ADN , Epigénesis Genética , Perfilación de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/genética , Linfoma de Células T/inmunología , Masculino , Células Tumorales CultivadasRESUMEN
Epigenetic therapy using histone deacetylase (HDAC) inhibitors has become an attractive project in new drug development. However, DNA methylation and histone acetylation are important epigenetic ways to regulate the occurrence and development of leukemia. Given previous studies, N-(2-aminophenyl)benzamide acridine (8a), as a histone deacetylase 1 (HDAC1) inhibitor, induces apoptosis and shows significant anti-proliferative activity against histiocytic lymphoma U937 cells. HDAC1 plays a role in the nucleus, which we confirmed by finding that 8a entered the nucleus. Subsequently, we verified that 8a mainly passes through the endogenous (mitochondrial) pathway to induce cell apoptosis. From the protein interaction data, we found that 8a also affected the expression of DNA methyltransferase 1 (DNMT1). Therefore, an experiment was performed to assess the binding of 8a to DNMT1 at the molecular and cellular levels. We found that the binding strength of 8a to DNMT1 enhanced in a dose-dependent manner. Additionally, 8a inhibits the expression of DNMT1 mRNA and its protein. These findings suggested that the anti-proliferative and pro-apoptotic activities of 8a against leukemia cells were achieved by targeting HDAC1 and DNMT1.
Asunto(s)
Apoptosis , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1/genética , Regulación Neoplásica de la Expresión Génica , Células HeLa , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Células K562 , Simulación del Acoplamiento Molecular , Neoplasias/enzimología , Neoplasias/fisiopatología , Células U937RESUMEN
Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.
Asunto(s)
Calcificación Fisiológica , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ligamento Periodontal/citología , Calcificación Fisiológica/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Osteogénesis/genética , Ftalimidas/farmacología , Triptófano/análogos & derivados , Triptófano/farmacología , Regulación hacia Arriba/efectos de los fármacos , Adulto JovenRESUMEN
Concomitant inhibition of key epigenetic pathways involved in silencing tumor suppressor genes has been recognized as a promising strategy for cancer therapy. Herein, we report a first-in-class series of quinoline-based analogues that simultaneously inhibit histone deacetylases (from a low nanomolar range) and DNA methyltransferase-1 (from a mid-nanomolar range, IC50 < 200 nM). Additionally, lysine methyltransferase G9a inhibitory activity is achieved (from a low nanomolar range) by introduction of a key lysine mimic group at the 7-position of the quinoline ring. The corresponding epigenetic functional cellular responses are observed: histone-3 acetylation, DNA hypomethylation, and decreased histone-3 methylation at lysine-9. These chemical probes, multitarget epigenetic inhibitors, were validated against the multiple myeloma cell line MM1.S, demonstrating promising in vitro activity of 12a (CM-444) with GI50 of 32 nM, an adequate therapeutic window (>1 log unit), and a suitable pharmacokinetic profile. In vivo, 12a achieved significant antitumor efficacy in a xenograft mouse model of human multiple myeloma.
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Antineoplásicos/química , Antineoplásicos/farmacología , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Antígenos de Histocompatibilidad/metabolismo , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
Epigenetic therapies may modulate the tumor microenvironment. We evaluated the safety and optimal sequence of combination DNA methyltransferase inhibitor guadecitabine with a granulocyte macrophage-colony-stimulating-factor (GM-CSF) secreting colon cancer (CRC) vaccine (GVAX) using a primary endpoint of change in CD45RO + T cells. 18 patients with advanced CRC enrolled, 11 underwent paired biopsies and were evaluable for the primary endpoint. No significant increase in CD45RO + cells was noted. Grade 3-4 toxicities were expected and manageable. Guadecitabine + GVAX was tolerable but demonstrated no significant immunologic activity in CRC. We report a novel trial design to efficiently evaluate investigational therapies with a primary pharmacodynamic endpoint.Trial registry Clinicaltrials.gov: NCT01966289. Registered 21 October, 2013.