Dataset Information

Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance.

ABSTRACT: The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors involved in the molecular response to hypoxia, to overcome GBM radioresistance. We used the U87 cell line subjected to chemical hypoxia generated by CoCl2 and exposed to 2 Gy of X-rays, as single or combined treatments, and evaluated gene expression changes of biomarkers involved in the Warburg effect, cell cycle control, and survival to identify the best molecular targets to be knocked-down, among those directly activated by the HIF-1α transcription factor. By this approach, glut-3 and pdk-1 genes were chosen, and the effects of their morpholino-induced gene silencing were evaluated by exploring the proliferative rates and the molecular modifications of the above-mentioned biomarkers. We found that, after combined treatments, glut-3 gene knockdown induced a greater decrease in cell proliferation, compared to pdk-1 gene knockdown and strong upregulation of glut-1 and ldha, as a sign of cell response to restore the anaerobic glycolysis pathway. Overall, glut-3 gene knockdown offered a better chance of controlling the anaerobic use of pyruvate and a better proliferation rate reduction, suggesting it is a suitable silencing target to overcome radioresistance.

SUBMITTER: Pucci G

PROVIDER: S-EPMC10889562 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance.

Pucci Gaia G Minafra Luigi L Bravatà Valentina V Calvaruso Marco M Turturici Giuseppina G Cammarata Francesco P FP Savoca Gaetano G Abbate Boris B Russo Giorgio G Cavalieri Vincenzo V Forte Giusi I GI

International journal of molecular sciences 20240208 4

The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors involved in the molecular response to hypoxia, to overcome GBM radioresistance. We used the U87 cell line subjected to chemical hypoxia generated by CoCl2 and exposed to 2 Gy of X-rays, as single or combined treatments, and evaluated gene expression changes of biomarkers involved in the Warburg effect, cell cycle control, and survi ...[more]

PMID: 38396757

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Project description:Hypoxia is a predominant feature in glioblastoma (GBM) and contributes greatly to its drug resistance. However, the molecular mechanisms which are responsible for the development of the resistant phenotype of GBM under hypoxic conditions remain unclear. To analyze the key pathways promoting therapy resistance in hypoxic GBM, we utilized the U87-MG cell line as a human GBM cell model and the human brain HEB cell line as a non-neoplastic brain cell model. These cell lines were cultured in the presence of 21, 5, and 1% O2 for 24 h. We detected the changes in transcriptional profiling and analyzed the biological processes and functional interactions for the genes with different expression levels under different hypoxia conditions. The results indicated that those alterations of U87-MG cells presented specific transcriptional signature in response to diverse hypoxia levels. Gene ontology analysis revealed that the genes related to the DNA replication and cell cycle were suppressed, while the genes involved in tissue and system development to promote cancer development were activated following hypoxia. Moreover, functional interaction analysis suggested that the epigenetic regulator HDAC3 and the transcriptional factors CEBPB and JUN played a central role in organ and system developmental process pathway. Previous studies reported the global alterations caused by activation of HDAC3, CEBPB, and JUN could form the molecular basis of the resistance to chemotherapy and radiation therapy of hypoxic GBM. In our study, the significant growth inhibitory effect of temozolomide on hypoxic GBM cells could be promoted under downregulation of these genes. The experiment suggested that HDAC3, CEBPB, and JUN were closely involved in the drug-resistance phenotype of hypoxic GBM. In summary, we profiled the hypoxia-dependent changes in the transcriptome of the U87-MG cell line and the human brain cell line HEB to identify the transcriptional signatures of U87-MG cells and elucidate the role of hypoxia in the drug-resistant phenotype of GBM. Furthermore, we identified three key genes and explored their important roles in the drug resistance of hypoxic GBM.

Project description:(1) Background: The sensitivity of head and neck squamous cell carcinoma (HNSCC) to ionizing radiation, among others, is determined by the number of cells with high clonogenic potential and stem-like features. These cellular characteristics are dynamically regulated in response to treatment and may lead to an enrichment of radioresistant cells with a cancer stem cell (CSC) phenotype. Epigenetic mechanisms, particularly DNA and histone methylation, are key regulators of gene-specific transcription and cellular plasticity. Therefore, we hypothesized that specific epigenetic targeting may prevent irradiation-induced plasticity and may sensitize HNSCC cells to radiotherapy. (2) Methods: We compared the DNA methylome and intracellular concentrations of tricarboxylic acid cycle metabolites in radioresistant FaDu and Cal33 cell lines with their parental controls, as well as aldehyde dehydrogenase (ALDH)-positive CSCs with negative controls. Moreover, we conducted a screen of a chemical library targeting enzymes involved in epigenetic regulation in combination with irradiation and analyzed the clonogenic potential, sphere formation, and DNA repair capacity to identify compounds with both radiosensitizing and CSC-targeting potential. (3) Results: We identified the histone demethylase inhibitor GSK-J1, which targets UTX (KDM6A) and JMJD3 (KDM6B), leading to increased H3K27 trimethylation, heterochromatin formation, and gene silencing. The clonogenic survival assay after siRNA-mediated knock-down of both genes radiosensitized Cal33 and SAS cell lines. Moreover, high KDM6A expression in tissue sections of patients with HNSCC was associated with improved locoregional control after primary (n = 137) and post-operative (n = 187) radio/chemotherapy. Conversely, high KDM6B expression was a prognostic factor for reduced overall survival. (4) Conclusions: Within this study, we investigated cellular and molecular mechanisms underlying irradiation-induced cellular plasticity, a key inducer of radioresistance, with a focus on epigenetic alterations. We identified UTX (KDM6A) as a putative prognostic and therapeutic target for HNSCC patients treated with radiotherapy.

Dataset Information

Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance.

Publications

Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance.

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