RESUMEN
BACKGROUND: The high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive. METHOD: Expression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS. RESULT: We determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model. CONCLUSION: These findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.
Asunto(s)
Células Madre Neoplásicas , Tolerancia a Radiación , Ubiquitina Tiolesterasa , Humanos , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Tolerancia a Radiación/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de la radiación , Animales , Ratones , Línea Celular Tumoral , Glioma/patología , Glioma/genética , Glioma/radioterapia , Glioma/metabolismo , Apoptosis/genética , Apoptosis/efectos de la radiación , Ubiquitinación , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Ratones Desnudos , Fenotipo , Regulación Neoplásica de la Expresión Génica , PronósticoRESUMEN
Mesenchymal glioma stem cells (MES GSCs) are a subpopulation of cells in glioblastoma (GBM) that contribute to a worse prognosis owing to their highly aggressive nature and resistance to radiation therapy. Here, OCT4 is characterized as a critical factor in sustaining the stemness phenotype of MES GSC. We find that OCT4 is expressed intensively in MES GSC and is intimately associated with poor prognosis, moreover, OCT4 depletion leads to diminished invasive capacity and impairment of the stem phenotype in MES GSC. Subsequently, we demonstrated that USP5 is a deubiquitinating enzyme which directly interacts with OCT4 and preserves OCT4 stability through its deubiquitination. USP5 was additionally proven to be aberrantly over-expressed in MES GSCs, and its depletion resulted in a noticeable diminution of OCT4 and consequently a reduced self-renewal and tumorigenic capacity of MES GSCs, which can be substantially restored by ectopic expression of OCT4. In addition, we detected the dominant molecule that regulates USP5 transcription, E2F1, with dual luciferase reporter gene analysis. In combination, targeting the E2F1-USP5-OCT4 axis is a potentially emerging strategy for the therapy of GBM.
Asunto(s)
Neoplasias Encefálicas , Factor de Transcripción E2F1 , Células Madre Neoplásicas , Factor 3 de Transcripción de Unión a Octámeros , Proteasas Ubiquitina-Específicas , Humanos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismo , Animales , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismo , Glioma/patología , Glioma/genética , Glioma/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Ratones , Estabilidad Proteica , Glioblastoma/patología , Glioblastoma/genética , Glioblastoma/metabolismo , UbiquitinaciónRESUMEN
This paper describes a method for determining ascorbic acid by the suppression of DTMC-H2O2 chemiluminescence. Various factors affecting the chemiluminescence determination of ascorbic acid were examined. Under the optimal conditions (1.2 x 10(-4) mol.L-1 of DTMC, 5.0 x 10(-5) mol.L-1 of H2O2, and pH 11.4 Na2HPO4-NaOH), the quenching of the chemiluminescent intensity was proportional to the concentration of ascorbic acid in the range of 1.0 x 10(-7)-8.0 x 10(-6) mol.L-1, and the detection limit was 8.0 x 10(-8) mol.L-1 (S/N = 3). The RSD was 4.6% for 1.0 x 10(-6) mol.L-1 of ascorbic acid (n = 10). The selectivity of this method was high and no making agent required. The proposed method has been applied to the determination of ascorbic acid in real samples. The obtained results were in agreement with those by CuSO4-KI titration.