RESUMEN
Purpose: DNA damage contributes to the pathogenesis of age-related cataract (ARC) and is repaired through the nucleotide excision repair (NER) pathway, which includes ERCC6. Evidence has demonstrated that defective autophagy leads to lens organelle degradation and cataract. This study aimed to investigate the effects of ERCC6 on autophagy and determine its mechanisms in ARC.Methods: The clinical case-control study comprised 30 patients with ARC and 30 age-matched controls who received transparent lens extraction. Transmission electron microscopy was used to assess the ultrastructure of autophagic vesicles in lens anterior capsule tissues and lens epithelial cell line (SRA01/04). Real-time polymerase chain reaction and western blot analyses were performed to measure relative gene expression levels. Gene expression levels and localization were assessed by immunofluorescence. A coimmunoprecipitation assay was used to investigate the relationship between CSB which encoded by ERCC6 and VCP. ERCC6-siRNA and let-7 c-5p mimic were used to alter the expression of ERCC6 and let-7 c-5p.Results: Autophagy induction occurred in lens anterior capsule tissues of patients with ARC and in UVB-induced SRA01/04 cells, where the number of LC3B puncta was increased. Consistent with this result, the expression of beclin1 (BECN1) and LC3B, in addition to that of p62, was increased. Additionally, ERCC6 expression decreased, and silencing ERCC6 induced increases in the expression of BECN1, LC3B and p62. Moreover, CSB interacted with VCP, and let-7 c-5p induced dysregulation of autophagy by targeting ERCC6.Conclusion: In ARC, Let-7 c-5p-mediated downregulation of ERCC6 might prevent the degradation of autophagic vacuoles. CSB binds to VCP, inducing autophagosomes to combine with lysosomes and be degraded.
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Cápsula Anterior del Cristalino/metabolismo , Catarata/genética , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteína que Contiene Valosina/metabolismo , Anciano , Cápsula Anterior del Cristalino/ultraestructura , Autofagia , Western Blotting , Estudios de Casos y Controles , Catarata/metabolismo , Catarata/patología , Línea Celular , ADN Helicasas/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Células Epiteliales/ultraestructura , Femenino , Humanos , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Proteínas de Unión a Poli-ADP-Ribosa/biosíntesisRESUMEN
Purpose: To explore the regulatory role of ERCC6 in the circRNA-miRNA-mRNA network using a cellular ERCC6 overexpression model (OE-ERCC6) in lens epithelial cells.Methods: The expression profiles of circRNAs, miRNAs and mRNAs were determined by RNA-seq, and a regulatory circRNA-miRNA-mRNA network was constructed via bioinformatics. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used for the functional annotation of circRNA host genes, differentially expressed (DE) genes, and miRNA targets.Results: The DE molecules between the OE-ERCC6 and control groups included 269 circRNAs, 241 miRNAs and 3500 mRNAs. We validated 5 selected DE reads of circRNAs (hsa_circ_0001009, hsa_circ_0002024, hsa_circ_0004592, hsa_circ_0001900 and hsa_circ_0001017). Subsequent bioinformatics analysis revealed that the DE circRNAs are mainly involved in oxidative stress- and cell death-related signaling pathways. Finally, a circRNA-miRNA-mRNA network focusing on DNA damage and cell death, which involved 5 circRNAs, 13 miRNAs and 107 mRNAs, was constructed.Conclusion: We constructed a circRNA-miRNA-mRNA network that is regulated by ERCC6. DE circRNAs have the potential to become therapeutic targets related to the lens lesions observed in ARC. The establishment of related in vivo and in vitro models could be a future direction to confirm these hypotheses.
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ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Cápsula del Cristalino/citología , MicroARNs/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Circular/genética , ARN Mensajero/genética , Western Blotting , Catarata/genética , Catarata/metabolismo , Catarata/patología , Células Cultivadas , ADN Helicasas/biosíntesis , Reparación del ADN , Enzimas Reparadoras del ADN/biosíntesis , Células Epiteliales/patología , Perfilación de la Expresión Génica/métodos , Humanos , Cápsula del Cristalino/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/biosíntesisRESUMEN
AIMS: The study focused on the expression and role of a recent potential cancer therapeutic target protein, MutT Homolog1 (MTH1). MTH1 gets activated in an increased reactive oxygen species (ROS) environment and removes the oxidized nucleotides from the cell. The study aimed to check the role of MTH1 in DNA damage and apoptosis, migration and angiogenesis and also to examine its regulation in glioma. MAIN METHODS: The experiments were carried out in human glioma tissue samples and brain tissues of epilepsy patients (non-tumor control). We used two human glioblastomas cell lines, U87MG and U251MG cells. In order to study the role of MTH1 in glioma and to analyze the relation of MTH1 with Hif1α, we have used MTH1 siRNA and Hif1α siRNA respectively. KEY FINDINGS: We found an increased expression of MTH1 in glioma tissues compared to the non-tumor brain tissues. Correlation analysis revealed that those samples showing reduced expression of MTH1 also had high levels of DNA damage and apoptotic markers, while diminished expression of angiogenesis regulators and levels of migration. MTH1 knockdown in vitro by siRNA in tumor cell lines corroborates the above observation. This justifies the emergence of MTH1 inhibitors as potential first-in-class drugs. Mechanistically, our observations suggest that Hif1α may modulate MTH1 expression. SIGNIFICANCE: We found elevated MTH1 expression in glioma irrespective of their grades, while its inhibition affects multiple tumor progression pathways, and that targeting Hif1α could simulate the same.
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Neoplasias Encefálicas/metabolismo , Enzimas Reparadoras del ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Monoéster Fosfórico Hidrolasas/biosíntesis , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Enzimas Reparadoras del ADN/genética , Glioma/genética , Glioma/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Clasificación del Tumor/métodos , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
Background: Glioblastoma is a malignant and very aggressive brain tumor with a poor prognosis. Despite having chemotherapy concomitant with surgery and/or radiation therapy, the median survival of glioblastoma-affected people is less than 1 year. Temozolomide (TMZ) is a chemotherapeutic used as a first line treatment of glioblastoma. Several studies have reported that resistance to TMZ due to overexpression of O6-methylguanine-DNA methyltransferase (MGMT) is the main reason for treatment failure. Several studies described that pulsed-electromagnetic field (EMF) exposure could induce cell death and influence gene expression. Materials and Methods: In this study the authors assessed the effects of EMF (50 Hz, 70 G) on cytotoxicity, cell migration, gene expression, and protein levels in TMZ-treated T98 and A172 cell lines. Results: In this study, the authors show that treatment with a combination of TMZ and EMF enhanced cell death and decreased the migration potential of T98 and A172 cells. The authors also observed overexpression of the p53 gene and downregulation of cyclin-D1 protein in comparison to controls. In addition, T98 cells expressed the MGMT protein following treatment, while the A172 cells did not express MGMT. Conclusion: Their data indicate that EMF exposure improved the cytotoxicity of TMZ on T98 and A172 cells and could partially affect resistance to TMZ in T98 cells.
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Neoplasias Encefálicas/terapia , Ciclina D2/biosíntesis , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Glioblastoma/terapia , Magnetoterapia , Temozolomida/farmacología , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas Supresoras de Tumor/biosíntesis , Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Ciclina D2/genética , Resistencia a Antineoplásicos/efectos de la radiación , Campos Electromagnéticos , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
A signalling pathway involving PLEKHG5 (guanine exchange factor) for the Ras superfamily member RAB26 to transcription factor NF-κB was discovered in autophagy. PLEKHG5 was reported in glioblastoma multiforme (GBM) and correlates with patient survival. Thus, the generation of a cellular model for understanding PLEKHG5 signalling is the study purpose. We generated a CRISPR/Cas9-mediated knockout of PLEKHG5 in U251-MG glioblastoma cells and analysed resulting changes. Next, we used a mRFP-GFP-LC3+ reporter for visualisation of autophagic defects and rescued the phenotype of PLEKHG5 wildtype via transduction of a constitutively active RAB26QL-plasmid. Effects of overexpressing RAB26 were investigated and correlated with the O6-methylguanine-DNA methyltransferase (MGMT) and cellular survival. PLEKHG5 knockout showed changes in morphology, loss of filopodia and higher population doubling times. Accumulation of autolysosomes was resulted by decreased LAMP-1 in PLEKHG5-deficient cells. Rescue of PLEKHG5-/- restored the downregulation of RhoA activity, showed faster response to tumour necrosis factor and better cellular fitness. MGMT expression was activated after RAB26 overexpression compared to non-transduced cells. Survival of PLEKHG5 knockout was rescued together with sensitivity to temozolomide by RAB26QL. This study provides new insights in the PLEKHG5/RAB26 signalling within U251-MG cells, which suggests potential therapeutic strategies in other glioma cells and further in primary GBM.
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Autofagia , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/biosíntesis , Línea Celular Tumoral , Supervivencia Celular , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Técnicas de Inactivación de Genes , Glioblastoma/genética , Glioblastoma/patología , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Proteínas Supresoras de Tumor/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Glioblastoma (GBM) is a common and aggressive brain tumor with a median survival of 12-15 months. Temozolomide (TMZ) is a first-line chemotherapeutic agent used in GBM therapy, but the occurrence of drug resistance limits its antitumor activity. The natural compound cedrol has remarkable antitumor activity and is derived from Cedrus atlantica. In this study, we investigated the combined effect of TMZ and cedrol in GBM cells in vitro and in vivo. The TMZ and cedrol combination treatment resulted in consistently higher suppression of cell proliferation via regulation of the AKT and MAPK signaling pathways in GBM cells. The combination treatment induced cell cycle arrest, cell apoptosis, and DNA damage better than either drug alone. Furthermore, cedrol reduced the expression of proteins associated with drug resistance, including O6-methlyguanine-DNA-methyltransferase (MGMT), multidrug resistance protein 1 (MDR1), and CD133 in TMZ-treated GBM cells. In the animal study, the combination treatment significantly suppressed tumor growth through the induction of cell apoptosis and decreased TMZ drug resistance. Moreover, cedrol-treated mice exhibited no significant differences in body weight and improved TMZ-induced liver damage. These results imply that cedrol may be a potential novel agent for combination treatment with TMZ for GBM therapy that deserves further investigation.
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Antineoplásicos Alquilantes/farmacología , Antineoplásicos Fitogénicos/farmacología , Daño del ADN , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Resistencia a Antineoplásicos/efectos de los fármacos , Sesquiterpenos Policíclicos/farmacología , Temozolomida/farmacología , Proteínas Supresoras de Tumor/biosíntesis , Animales , Antineoplásicos Alquilantes/toxicidad , Apoptosis/efectos de los fármacos , Cedrus/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Sinergismo Farmacológico , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Estructura Molecular , Proteína Oncogénica v-akt/efectos de los fármacos , Temozolomida/toxicidad , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pancreatic cancer is a malignant cancer with poor prognosis. This study aimed to explore how O6-methylguanine-DNA methyltransferase (MGMT) affects the gemcitabine resistance of pancreatic cancer cells by the regulatory role of SHH/GLI signaling pathway. MGMT inhibition induced by lomeguatrib (LM) suppressed the proliferation, invasion, migration and autophagy, promoted the apoptosis of PanC-1/GEM cells and up-regulated the GEM inhibition rates for PanC-1/GEM cells. Moreover, MGMT inhibition increased the expression of Caspase-3 and Bax and decreased the expression of Bcl-2, Beclin1 and Atg5 in PanC-1/GEM cells. PVT1 silencing could also produce the similar effects of MGMT inhibition induced by LM on PanC-1/GEM cells. And, PVT1 silencing could inhibit the SHH/GLI signaling pathway in PanC-1/GEM cells by regulating the MGMT expression. miR-409 was demonstrated to be a potential target of PVT1 and SHH was demonstrated to be a potential target of miR-409. Furthermore, GLI overexpression could reverse the effects of PVT1 silencing. In the xenograft model of pancreatic cancer, nude mice were treated with GEM. MGMT inhibition suppressed the tumor growth and autophagy and promoted the apoptosis in tumor tissues. And, PVT1 silencing could inhibit the SHH/GLI signaling pathway in tumor tissues. In conclusion, MGMT inhibition could suppress the proliferation, invasion, migration and autophagy and promote the apoptosis of PanC-1/GEM cells in vitro and in vivo. PVT1 silencing may affect the PanC-1/GEM cells through changing the MGMT expression by inhibiting the SHH/GLI signaling pathway.
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Antimetabolitos Antineoplásicos/farmacología , Metilasas de Modificación del ADN/biosíntesis , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/genética , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Animales , Autofagia/efectos de los fármacos , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Desoxicitidina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Neoplasias Pancreáticas/patología , Purinas/farmacología , Purinas/uso terapéutico , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
It has been observed that some patients with colorectal cancer due to germline or double somatic pathogenic variants in the mismatch repair (MMR) genes may have intact protein expression in their tumors as assessed by immunohistochemistry (IHC). This has been speculated to occur more frequently in Lynch syndrome (LS) cases due to pathogenic missense mutations, leading to expression of a full-length but nonfunctional protein with retained antigenicity. Our goals were to study the frequency of unexpected MMR expression in colorectal cancers among LS cases with missense mutations, LS cases with truncating mutations, as well as cases with double somatic MMR mutations and evaluate if the unexpected MMR expression is more common in certain categories. IHC slides were available for 82 patients with MMR deficiency without methylation, which included 56 LS cases and 26 double somatic MMR mutation cases. Sixteen of 82 MMR-defective cases showed unexpected MMR expression, with 10 cases showing tumor staining weaker than the control and 6 cases (7%) showing intact staining. Unexpected MMR expression was most commonly seen with LS cases with missense mutations (4 of 9, 44%), followed by MMR double somatic mutation cases (7 of 26, 27%), and finally by LS cases with truncating mutations (5 of 47, 11%). Cautious interpretation of MMR IHC is advised when dealing with tumor staining that is weaker than the control regardless of the percentage of tumor staining as these cases may harbor pathogenic MMR gene mutations. Missense mutations may account for some LS cases that may be missed by IHC alone. Strict adherence to proper interpretation of IHC with attention to staining intensity and the status of heterodimer partner protein will prevent many potential misses.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Enzimas Reparadoras del ADN/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Enzimas Reparadoras del ADN/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Adulto JovenRESUMEN
In the present study, to delve into the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with temozolomide (TMZ) on high-grade glioma cells and related mechanism, six cases of high-grade glioma cells from patient's tumor tissues were cultured. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was performed to detect cell proliferation and toxicity. Flow cytometry was performed to ascertain cell cycle and apoptosis rate. To detect the expressions of O6-methylguanine-DNA methyltransferase (MGMT) methylation status and MGMT protein, respectively, specific PCR and immunofluorescence were performed. According to the results of MTT assay, compared with the results of control group, GM-CSF group exhibited enhanced cell viability in varying degrees. In three cases of cells (MGMT gene methylation), the combination group [(67.67 ± 1.16), (68.13 ± 1.06), (68.42 ± 1.73)] had noticeably lower cell viability than the corresponding TMZ group [(90.00 ± 1.73), (82.33 ± 1.53), (82.67 ± 2.11)] (P < 0.01). Nevertheless, the two groups showed no significant difference in another three cases (MGMT gene unmethylated) (P > 0.05). In combination group, the apoptosis rate of the MGMT methylation cells was higher than that in the corresponding TMZ group (P < 0.01), which is consistent with MTT assay results. In all six cases of primary glioma cells, the fraction of cells in G1 phase of GM-CSF-treated group was noticeably down-regulated and was up-regulated in S phase (P < 0.01). GM-CSF could induce high-grade glioma cells to rapidly enter the cell cycle, thereby enhancing the lethal effect of TMZ on glioma cells with MGMT gene promoter methylation. However, this effect is not ideal on glioma cells with MGMT unmethylation.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Astrocitoma/tratamiento farmacológico , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Temozolomida/farmacología , Astrocitoma/enzimología , Astrocitoma/genética , Astrocitoma/patología , Ciclo Celular/efectos de los fármacos , Neoplasias del Sistema Nervioso Central/enzimología , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Metilación de ADN , Metilasas de Modificación del ADN/biosíntesis , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/genética , Sinergismo Farmacológico , Glioblastoma/enzimología , Glioblastoma/genética , Glioblastoma/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Humanos , Inmunohistoquímica , Clasificación del Tumor , Regiones Promotoras Genéticas , Temozolomida/administración & dosificación , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
We investigated the prognostic significance of Nudix hydrolase 1 (NUDT1) in hepatocellular carcinoma (HCC). NUDT1 mRNA and protein levels were significantly higher in HCC tissues than normal liver tissues. The level of NUDT1 expression correlated with tumor grade, stage, size, differentiation, degree of vascular invasion, overall survival (OS), and disease-free survival (DFS) in HCC patients. Multivariate analysis showed that NUDT1 expression was an independent prognostic factor for OS and DFS in HCC patients. We constructed a prognostic nomogram with NUDT1 expression, AFP levels, vascular invasion, Child-Pugh classification, age, sex, AJCC staging, and tumor differentiation as variables. This nomogram was highly accurate in predicting the 5-year OS of HCC patients (c-index= 0.709; AUC= 0.740). NUDT1 silencing in HCC cells significantly reduced their survival, colony formation, migration, and invasiveness. Gene set enrichment analysis showed that biological pathways related to cell cycle, fatty acid metabolism, bile acid and bile salt metabolism, and PLK1 signaling were associated with NUDT1, as were the gene ontology terms "DNA binding transcription activator activity," "RNA polymerase II," "nuclear division," and "transmembrane transporter activity." Our study thus demonstrates that NUDT1 is a prognostic biomarker with therapeutic potential in HCC patients.
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Carcinoma Hepatocelular/genética , Enzimas Reparadoras del ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Monoéster Fosfórico Hidrolasas/genética , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Enzimas Reparadoras del ADN/biosíntesis , ADN de Neoplasias/genética , Femenino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Monoéster Fosfórico Hidrolasas/biosíntesis , Pronóstico , Regulación hacia ArribaRESUMEN
OBJECTIVE: Glutamatergic signaling significantly promotes proliferation, migration, and invasion in glioblastoma (GBM). Riluzole, a metabotropic glutamate receptor 1 inhibitor, reportedly suppresses GBM growth. However, the effects of combining riluzole with the primary GBM chemotherapeutic agent, temozolomide (TMZ), are unknown. This study aimed to investigate the efficacy of combinatorial therapy with TMZ/riluzole for GBM in vitro and in vivo. METHODS: Three GBM cell lines, T98G (human; O6-methylguanine DNA methyltransferase [MGMT] positive), U87MG (human; MGMT negative), and GL261 (murine; MGMT positive), were treated with TMZ, riluzole, or a combination of both. The authors performed cell viability assays, followed by isobologram analysis, to evaluate the effects of combinatorial treatment for each GBM cell line. They tested the effect of riluzole on MGMT, a DNA repair enzyme causing chemoresistance to TMZ, through quantitative real-time reverse transcription polymerase chain reaction in T98G cells. Furthermore, they evaluated the efficacy of combinatorial TMZ/riluzole treatment in an orthotopic mouse allograft model of MGMT-positive GBM using C57BL/6 J mice and GL261 cells. RESULTS: Riluzole displayed significant time- and dose-dependent growth-inhibitory effects on all GBM cell lines assessed independently. Riluzole enhanced the antitumor effect of TMZ synergistically in MGMT-positive but not in MGMT-negative GBM cell lines. Riluzole singularly suppressed MGMT expression, and it significantly suppressed TMZ-induced MGMT upregulation (p < 0.01). Furthermore, combinatorial TMZ/riluzole treatment significantly suppressed tumor growth in the intracranial MGMT-positive GBM model (p < 0.05). CONCLUSIONS: Riluzole attenuates TMZ-induced MGMT upregulation and enhances the antitumor effect of TMZ in MGMT-positive GBMs. Therefore, combinatorial TMZ/riluzole treatment is a potentially promising novel therapeutic regimen for MGMT-positive GBMs.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Glioblastoma/tratamiento farmacológico , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Animales , Antineoplásicos Alquilantes/administración & dosificación , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Metilasas de Modificación del ADN/biosíntesis , Metilasas de Modificación del ADN/genética , Reparación del ADN/efectos de los fármacos , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/genética , Sinergismo Farmacológico , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Riluzol/administración & dosificación , Temozolomida/administración & dosificación , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Our previous study revealed a higher incidence of gene dynamic mutation in newborns conceived by IVF, highlighting that IVF may be disruptive to the DNA stability of IVF offspring. However, the underlying mechanisms remain unclear. The DNA damage repair system plays an essential role in gene dynamic mutation and neurodegenerative disease. To evaluate the long-term impact of IVF on DNA damage repair genes, we established an IVF mouse model and analyzed gene and protein expression levels of MSH2, MSH3, MSH6, MLH1, PMS2, OGG1, APEX1, XPA and RPA1 and also the amount of H2AX phosphorylation of serine 139 which is highly suggestive of DNA double-strand break (γH2AX expression level) in the brain tissue of IVF conceived mice and their DNA methylation status using quantitative real-time PCR, western blotting and pyrosequencing. Furthermore, we assessed the capacity of two specific non-physiological factors in IVF procedures during preimplantation development. The results demonstrated that the expression and methylation levels of some DNA damage repair genes in the brain tissue of IVF mice were significantly changed at 3 weeks, 10 weeks and 1.5 years of age, when compared with the in vivo control group. In support of mouse model findings, oxygen concentration of in vitro culture environment was shown to have the capacity to modulate gene expression and DNA methylation levels of some DNA damage repair genes. In summary, our study indicated that IVF could bring about long-term alterations of gene and protein expression and DNA methylation levels of some DNA damage repair genes in the brain tissue and these alterations might be resulted from the different oxygen concentration of culture environment, providing valuable perspectives to improve the safety and efficiency of IVF at early embryonic stage and also throughout different life stages.
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Encéfalo/metabolismo , Daño del ADN , Enzimas Reparadoras del ADN/biosíntesis , Reparación del ADN/genética , Fertilización In Vitro , Proteínas del Tejido Nervioso/biosíntesis , Animales , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Encéfalo/embriología , Encéfalo/enzimología , Metilación de ADN , Enzimas Reparadoras del ADN/genética , Transferencia de Embrión , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Estradiol/farmacología , Femenino , Fertilización In Vitro/efectos adversos , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteínas del Tejido Nervioso/genética , Recuperación del Oocito , Oxígeno/farmacologíaRESUMEN
INTRODUCTION: Transsphenoidal surgery is the treatment of choice in Cushing's disease (CD), although even late recurrences occur in some patients. Low expression of O-6-methylguanine-DNA methyltransferase (MGMT) has been linked to a high risk of relapse in pituitary tumours, but the evidence for corticotroph adenomas is limited. Therefore, we investigated whether MGMT expression was associated with CD remission or clinicopathological markers of tumour aggressiveness among patients with corticotroph adenomas. MATERIAL AND METHODS: We included 72 consecutive patients (83% female, mean age ±SD: 44.15 ±15.15 years) with CD, who underwent transsphenoidal adenomectomy between 2012 and 2018. The invasiveness of corticotroph tumours was assessed based on the Knosp scale. Immunohistochemistry was used to analyse MGMT expression as well as the proliferation markers (Ki-67, p53, mitotic index). Electron microscopy was used to categorise tumours into densely or sparsely granulated. Early biochemical remission was evaluated in all patients 6 months after pituitary surgery. RESULTS: Early remission was observed in 47 (65%) patients 6 months after surgery. MGMT expression was > 75% in half of all tumours, < 25% in 14 tumours, and 25-50% or 50-75% in 11 tumours. Lower MGMT expression was associated with a larger tumour diameter (p = 0.001), higher adrenocorticotropic hormone (ACTH) concentration (p = 0.002), higher p53 expression (p = 0.026), and higher frequency of sparsely granulated corticotroph adenomas (p = 0.009). Low MGMT expression was significantly related to lower frequency of early clinical remission (p = 0.005). CONCLUSIONS: MGMT predicted the outcomes of transsphenoidal surgery for CD. Pituitary corticotroph adenomas with low MGMT expression may be associated with increased invasiveness and poorer prognosis.
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Adenoma Hipofisario Secretor de ACTH/patología , Adenoma/patología , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Adenoma Hipofisario Secretor de ACTH/metabolismo , Adenoma Hipofisario Secretor de ACTH/ultraestructura , Adenoma/metabolismo , Adenoma/ultraestructura , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/etiologíaRESUMEN
Immunohistochemistry for mismatch repair protein expression is widely used as a surrogate for microsatellite instability status-an important signature for immunotherapy and germline testing. There are no systematic analyses examining the sensitivity of immunohistochemistry for microsatellite instability-high status. Mismatch repair immunohistochemistry and microsatellite instability testing were performed routinely as clinically validated assays. We classified germline/somatic mutation types as truncating (nonsense, frameshift, and in/del) versus missense and predicted pathogenicity of the latter. Discordant cases were compared with concordant groups: microsatellite instability-high/mismatch repair-deficient for mutation comparison and microsatellite stable/mismatch repair-proficient for immunohistochemical comparison. 32 of 443 (7%) microsatellite instability-high cases had immunohistochemistry. Four additional microsatellite instability-high research cases had discordant immunohistochemistry. Of 36 microsatellite instability-high cases with discordant immunohistochemistry, 30 were mismatch repair-proficient, while six (five MLH1 and one MSH2) retained expression of the defective mismatch repair protein and lost its partner. In microsatellite instability-high tumors with discordant immunohistochemistry, we observed an enrichment in deleterious missense mutations over truncating mutations, with 69% (25/36) of cases having pathogenic germline or somatic missense mutations, as opposed to only 19% (7/36) in a matched microsatellite instability-high group with concordant immunohistochemistry (p = 0.0007). In microsatellite instability-high cases with discordant immunohistochemistry and MLH1 or PMS2 abnormalities, less cells showed expression (p = 0.015 and p = 0.00095, respectively) compared with microsatellite stable/mismatch repair-proficient cases. Tumor mutation burden, MSIsensor score, and truncating mismatch repair gene mutations were similar between microsatellite instability-high cases with concordant versus discordant immunohistochemical expression. Approximately 6% of microsatellite instability-high cases have retained mismatch repair protein expression and would be missed by immunohistochemistry-based testing, hindering patient access to immunotherapy. Another 1% of microsatellite instability-high cases show isolated loss of the defective gene's dimerization partner, which may lead to germline testing of the wrong gene. These cases are enriched for pathogenic mismatch repair missense mutations.
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Biomarcadores de Tumor/análisis , Reparación de la Incompatibilidad de ADN/genética , Enzimas Reparadoras del ADN/análisis , Neoplasias/genética , Biomarcadores de Tumor/genética , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/genética , Humanos , Inmunohistoquímica/métodos , Inestabilidad de Microsatélites , Mutación MissenseRESUMEN
The molecular mechanisms underlying the formation of nonfunctioning pituitary adenomas (NFAs) are largely unknown. In this study, we aimed to understand the relationship between NFAs and functional pituitary adenomas and the possible role of proteins involved in cell cycle, senescence, and DNA damage control mechanisms in the etiology of NFA. We analyzed pATM-S1981, pRb-S608, Rb, pE2F1-S364, p16, E2F1, p73, cyclin D1, and CHEK2 protein expression (in a group of 20 patients with acromegaly, 18 patients with Cushing's disease (CD), and 29 NFA patients) by immunohistochemistry and their relevant mRNA expression by qRT-PCR (in a group of 7 patients with acromegaly, 7 patients with CD, and 7 NFA patients). The clinical and histopathological results on the patients were statistically evaluated. pE2F1-S364 protein expression in the CD group was significantly lower than that in the NFA and acromegaly groups (p = 0.025, p = 0.034, respectively). However, the expression of the p16 protein was lower than in the NFA group than in the CD and acromegaly groups (p = 0.030, p = 0.033, respectively), and E2F1 protein expression was significantly higher in the NFA group than in the CD group (p = 0.025). p73 protein expression in patients with acromegaly was significantly higher (p = 0.031) than that in the CD group. CHEK2 mRNA expression in the CD group was significantly higher than that in the acromegaly group (p = 0.012). The selective and tumor-specific associations between E2F1, pE2F1-S364, CHEK2, and p73 mRNA and protein levels indicate their involvement in pituitary adenoma formation in NFA, CD, and acromegaly patients.
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Adenoma/metabolismo , Biomarcadores de Tumor/análisis , Proteínas de Ciclo Celular/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Neoplasias Hipofisarias/metabolismo , Adenoma Hipofisario Secretor de ACTH/metabolismo , Femenino , Adenoma Hipofisario Secretor de Hormona del Crecimiento/metabolismo , Humanos , Masculino , Estudios RetrospectivosRESUMEN
Aberrant patterns in promoter methylation of tumor-suppressor genes and posttranslational modifications of histone proteins are considered as major features of malignancy. In this study, we aimed to investigate promoter methylation of three tumor-suppressor genes (BRCA-1, MGMT, and P16) and three histone marks (H3K9ac, H3K18ac, and H4K20me3) in patients with breast tumors. This case-control study included 27 patients with malignant breast tumors (MBT) and 31 patients with benign breast tumors (BBT). The methylation-specific PCR was used for determining promoter methylation of BRCA-1, MGMT, and P16 genes. Western blot analysis was performed to detect histone lysine acetylation (H3K9ac and H3K18ac) and lysine methylation (H4K20me3). BRCA-1 promoter methylation was detected in 44.4% of the MBT whereas this alteration was found in 9.7% of BBT (P = 0.005). The Kaplan-Meier analysis indicated that hypermethylation in BRCA-1 promoter was significantly associated with poor overall survival of patients with breast cancer (P = 0.039). MGMT promoter methylation was identified in 18.5% of MBT and 0.0% of the BBT (P = 0.01). The frequency of P16 promoter methylation was 25.8% in BBT and 11.1% in MBT (P = 0.12). As compared with BBT, MBT samples displayed the aberrant patterns of histones marks with hypomethylation of H4K20 and hypoacetylation of H3K18 (P = 0.03 and P = 0.04, respectively). There was a negative significant correlation between H3K9ac levels and tumor size in MBT group (r = -0.672; P = 0.008). The present findings suggest that promoter hypermethylation of MGMT and BRCA-1 genes along with alterations in H3K18ac and H4K20me3 levels may have prognostic values in patients with breast cancer. Moreover, the detection of these epigenetic modifications in breast tumors could be helpful in finding new methods for breast cancer therapy.
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Proteína BRCA1/biosíntesis , Neoplasias de la Mama/metabolismo , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Adulto , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Metilación de ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Femenino , Histonas/genética , Humanos , Persona de Mediana Edad , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/genéticaRESUMEN
Chemoresistance is the primary obstacle in the treatment of locally advanced and metastatic nasopharyngeal carcinoma (NPC). Recent evidence suggests that the transcription factor forkhead box M1 (FoxM1) is involved in chemoresistance. Our group previously confirmed that FoxM1 is overexpressed in NPC. In this study, we investigated the role of FoxM1 in cisplatin resistance of the cell lines 5-8F and HONE-1 and explored its possible mechanism. Our results showed that FoxM1 and NBS1 were both overexpressed in NPC tissues based on data from the GSE cohort (GSE12452). Then, we measured FoxM1 levels in NPC cells and found FoxM1 was overexpressed in NPC cell lines and could be stimulated by cisplatin. MTT and clonogenic assays, flow cytometry, γH2AX immunofluorescence, qRT-PCR, and western blotting revealed that downregulation of FoxM1 sensitized NPC cells to cisplatin and reduced the repair of cisplatin-induced DNA double-strand breaks via inhibition of the MRN (MRE11-RAD50-NBS1)-ATM axis, which might be related to the ability of FoxM1 to regulate NBS1. Subsequently, we demonstrated that enhanced sensitivity of FoxM1 knockdown cells could be reduced by overexpression of NBS1. Taken together, our data demonstrate that downregulation of FoxM1 could improve the sensitivity of NPC cells to cisplatin through inhibition of MRN-ATM-mediated DNA repair, which could be related to FoxM1-dependent regulation of NBS1. [BMB Reports 2019; 52(3): 208-213].
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Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Cisplatino/farmacología , Reparación del ADN , Proteína Forkhead Box M1/genética , Carcinoma Nasofaríngeo/tratamiento farmacológico , Carcinoma Nasofaríngeo/metabolismo , Ácido Anhídrido Hidrolasas , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/biosíntesis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Resistencia a Antineoplásicos , Proteína Forkhead Box M1/biosíntesis , Proteína Forkhead Box M1/metabolismo , Humanos , Proteína Homóloga de MRE11/antagonistas & inhibidores , Proteína Homóloga de MRE11/biosíntesis , Proteína Homóloga de MRE11/genética , Carcinoma Nasofaríngeo/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Defects in Cockayne syndrome type A (CSA), a gene involved in nucleotide excision repair, cause an autosomal recessive syndrome characterized by growth failure, progressive neurological dysfunction, premature aging, and skin photosensitivity and atrophy. Beyond its role in DNA repair, the CSA protein has additional functions in transcription and oxidative stress response, which are not yet fully elucidated. Here, we investigated the role of CSA protein in primary human keratinocyte senescence. Primary keratinocytes from three patients with CS-A displayed premature aging features, namely premature clonal conversion, high steady-state levels of reactive oxygen species and 8-OH-hydroxyguanine, and senescence-associated secretory phenotype. Stable transduction of CS-A keratinocytes with the wild-type CSA gene restored the normal cellular sensitivity to UV irradiation and normal 8-OH-hydroxyguanine levels. Gene correction was also characterized by proper restoration of keratinocyte clonogenic capacity and expression of clonal conversion key regulators (p16 and p63), decreased NF-κB activity and, in turn, the expression of its targets (NOX1 and MnSOD), and the secretion of senescence-associated secretory phenotype mediators. Overall, the CSA protein plays an important role in protecting cells from senescence by facilitating DNA damage processing, maintaining physiological redox status and keratinocyte clonogenic ability, and reducing the senescence-associated secretory phenotype-mediated inflammatory phenotype.
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Síndrome de Cockayne/genética , Enzimas Reparadoras del ADN/genética , ADN/genética , Regulación de la Expresión Génica , Queratinocitos/metabolismo , Estrés Oxidativo , Envejecimiento de la Piel/genética , Factores de Transcripción/genética , Células Cultivadas , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patología , Daño del ADN , Reparación del ADN , Enzimas Reparadoras del ADN/biosíntesis , Humanos , Queratinocitos/patología , Factores de Transcripción/biosíntesisRESUMEN
BACKGROUND Astrocytomas are the most common primary brain neoplasms. Biological indicators of astrocytomas can reflect its biological characteristics. The aim of this study was to assess the expression of the pathological glial fibrillary acidic protein (GFAP) Topo IIα and O6-methylguanine-DNA methyltransferase (MGMT) in astrocytomas using magnetic resonance (MR) diffusion kurtosis imaging (DKI) to evaluate the biological characteristics of astrocytomas. MATERIAL AND METHODS Sixty-six patients with pathologically proven astrocytomas were enrolled in this study. All patients underwent conventional MRI head scanning, DKI scanning, and enhanced scanning under the same conditions. Spearman's rank correlation analysis and Bonferroni correction were used to compare the values of DKI and the expression levels of GFAP, Topo IIα, and MGMT between the 2 groups. RESULTS Mean kurtosis (MK) values were negatively correlated with the expression of GFAP (r=-0.836; P=0.03). However, these were positively correlated with the expression of Topo IIα (r=0.896; P=0.01). Moreover, fractional anisotropy (FA) values were not correlated with the expression of GFAP (r=0.366; P=0.05), Topo IIα (r=-0.562; P=0.05), or MGMT (r=-0.153; P=0.10). CONCLUSIONS MK was significantly associated with the expression of GFAP and Topo IIα. To a certain extent, applying DKI may show the biological behavior of tumor cell differentiation, proliferation activity, invasion, and metastasis, and guide individual treatment.
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Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , ADN-Topoisomerasas de Tipo II/biosíntesis , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteínas de Unión a Poli-ADP-Ribosa/biosíntesis , Proteínas Supresoras de Tumor/biosíntesis , Adulto , Anciano , Anisotropía , Astrocitoma/diagnóstico por imagen , Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Imagen de Difusión por Resonancia Magnética/métodos , Imagen de Difusión Tensora/métodos , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
OBJECTIVE: There is uncertainty about the prognostic significance of mismatch repair (MMR) deficiency in endometrial cancer. The objective was to evaluate clinical characteristics and outcomes of endometrial cancers based on MMR status within a population-based study. METHODS: This was a retrospective population-based cohort study of all endometrial cancer cases from the Vancouver Coastal Health Authority region, evaluated for 4 MMR proteins using immunohistochemistry from 2012 to 2015. Patients were classified as MMR deficient (dMMR, any MMR protein absent) or MMR proficient (pMMR), Demographics, tumor characteristics, recurrences, and survival rates were compared according to MMR status. RESULTS: There were 892 patients, with 650 pMMR (72.5%) and 242 dMMR tumors. The dMMR group had more endometrioid tumors (87.6% vs 74.0%, P < 0.001), lymphovascular space invasion (43.8% vs 30.8%, P = 0.001), and dedifferentiation (5.9% vs 1.5%, P < 0.001), but fewer grade 1 tumors compared with the pMMR group (31.8% vs 40.8%, P < 0.001). Median progression-free survival and overall survival have not been reached. After a median follow-up of 31 months (1-99 months), there was no difference in progression or recurrence rates between pMMR and dMMR tumors (19.5% vs 16.5%; P = 0.31). However, among those with nonendometrioid tumors, recurrence and mortality rates were significantly higher for pMMR than dMMR tumors (42.0% vs 10.0%, P = 0.001, and 36.1% vs 13.1%, P = 0.01, respectively), despite similar stage and lymphovascular space invasion distributions. DISCUSSION: In this population-based study, there were no significant differences in recurrence or survival outcomes according to MMR status in endometrial cancer. However, among those with nonendometrioid tumors, there were lower recurrence and mortality rates associated with MMR-deficient compared with MMR-proficient tumors.