Evaluation of the effects of simulated hypoxia by CoCl2 on radioresistance and change of hypoxia-inducible factors in human glioblastoma U87 tumor cell line

Purpose: Glioblastoma (GBM) is considered the most common and lethal type of brain tumor with a poor prognosis. GBM treatment has challenges due to its aggressive nature, which often causes treatment failure and recurrence. Hypoxia is one of the characteristics of glioblastoma tumors that contribute to radioresistance and malignant phenotypes of GBM. In this study, we aimed to determine the effects of hypoxia on the radiosensitivity of U87 GBM cells by the hypoxia-mimicking model. Methods: Following the treatment of cells with different concentrations of CoCl2, an MTT assay was used to evaluate the cytotoxicity of CoCl2. To understand the effects of Ionizing radiation on CoCl2-treated groups, cells were exposed to irradiation after pretreating with 100 mu M CoCl2, and a clonogenic survival assay was performed to determine the radiosensitivity of U87 cells. Also, the intracellular Reactive oxygen level was measured by 2 ',7 '-dichlorofluorescein diacetate (DCFDA) probe staining. Additionally, the expression of hypoxia-associated genes, including HIF-1 alpha, HIF-2 alpha, and their target genes (GLUT-1), was monitored by reverse transcription polymerase chain reaction (RT-PCR). Results: Our study revealed that the cell viability of CoCl2-treated cells was decreased in a concentrationdependent manner. Also, CoCl2 did not cause any cytotoxicity on U87 cells at a concentration of 100 mu M after treatment for 24 h. Colony formation assay showed that CoCl2 pretreatment induced radioresistance of tumor cells compared to non-treated cells. Also, CoCl2 can protect cells against irradiation by the clearance of ROS. Moreover, Real-time results showed that the mRNA expression of HIF-1 alpha and GLUT-1 were significantly upregulated following hypoxia induction and/or irradiation condition. However, the level of HIF-2 alpha mRNA did not change significantly in hypoxia or irradiation alone conditions, but it increased significantly only in hypoxia + irradiation conditions. Conclusion: Taken together, our results indicated that simulating hypoxia by CoCl2 can effectively increase hypoxia-associated genes, specially HIF-1 alpha and GLUT-1, but did not affect HIF-2 alpha gene expression. Also, it can increase the clearance of ROS, respectively, and it leads to inducing radioresistance of U87 cells.