Abstract
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.
Highlights
Glioblastoma (GBM) is the most common malignant and aggressive primary brain tumor in humans [1]
Hypoxia did not induce apoptosis of U87-MG cells. In contrast to these findings, HEB cells presented a higher apoptotic rate in the presence of 1% O2 compared with that noted under normoxic conditions (Figure 1B). These results revealed that 1% O2 hypoxia could inhibit proliferation and promote apoptosis of normal cells as opposed to GBM cells
The results of the flow cytometry assays indicated that treatment of U87-MG cells with 1% O2 increased the proportions of the cells at the G1 phase and decreased the percentage of the cells at the S phase compared with the corresponding percentages of the cells cultured in the presence of 21% O2 (Figure 1C), which indicated that hypoxia could induce G1 arrest of GBM cells
Summary
Glioblastoma (GBM) is the most common malignant and aggressive primary brain tumor in humans [1]. Liang [12] reported that hypoxic exposure could significantly increase the drug resistance of glioma cells. This property indicated no correlation with the expression of the multidrug resistance genes (MDR1, MRP, 06MT, and ERCC), which implied that other mechanisms might be acting in these hypoxic tumors [12]. The therapeutic strategies targeting HIF-1α are not able to eradicate tumors selectively [17], since HIFs participate in several physiological activities [18]. This suggests that a novel mechanism should be explored that defines the GBM drug-resistance phenotype under hypoxic conditions. The comprehensive understanding of the response of GBM to hypoxia will aid the identification of the efficient agents for GBM treatment
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