Elham Khakshour , Mohammad Taghi Bahreyni-Toossi , Kazem Anvari , Mohammad Amin Shahram , Fereshteh Vaziri-Nezamdoust , Hosein Azimian
{"title":"评估CoCl2模拟缺氧对人胶质母细胞瘤U87肿瘤细胞株放射抗性和缺氧诱导因子变化的影响","authors":"Elham Khakshour , Mohammad Taghi Bahreyni-Toossi , Kazem Anvari , Mohammad Amin Shahram , Fereshteh Vaziri-Nezamdoust , Hosein Azimian","doi":"10.1016/j.mrfmmm.2023.111848","DOIUrl":null,"url":null,"abstract":"<div><h3>Purpose</h3><p>Glioblastoma<span><span> (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 </span>radioresistance<span> 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.</span></span></p></div><div><h3>Methods</h3><p>Following the treatment of cells with different concentrations of CoCl<sub>2</sub><span>, an MTT assay was used to evaluate the cytotoxicity of CoCl</span><sub>2</sub><span>. To understand the effects of Ionizing radiation on CoCl</span><sub>2</sub>-treated groups, cells were exposed to irradiation after pretreating with 100 μM CoCl<sub>2,</sub><span> 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<span> (DCFDA) probe staining. Additionally, the expression of hypoxia-associated genes, including HIF-1α, HIF-2α, and their target genes (GLUT-1), was monitored by reverse transcription polymerase chain reaction (RT-PCR).</span></span></p></div><div><h3>Results</h3><p><span>Our study revealed that the cell viability of CoCl</span><sub>2</sub>-treated cells was decreased in a concentration-dependent manner. Also, CoCl<sub>2</sub> did not cause any cytotoxicity on U87 cells at a concentration of 100 μM after treatment for 24 h. Colony formation assay showed that CoCl<sub>2</sub><span> pretreatment induced radioresistance of tumor cells compared to non-treated cells. Also, CoCl</span><sub>2</sub> can protect cells against irradiation by the clearance of ROS. Moreover, Real-time results showed that the mRNA expression of HIF-1α and GLUT-1 were significantly upregulated following hypoxia induction and/or irradiation condition. However, the level of HIF-2α mRNA did not change significantly in hypoxia or irradiation alone conditions, but it increased significantly only in hypoxia + irradiation conditions.</p></div><div><h3>Conclusion</h3><p>Taken together, our results indicated that simulating hypoxia by CoCl<sub>2</sub> can effectively increase hypoxia-associated genes, specially HIF-1α and GLUT-1, but did not affect HIF-2α gene expression. Also, it can increase the clearance of ROS, respectively, and it leads to inducing radioresistance of U87 cells.</p></div>","PeriodicalId":49790,"journal":{"name":"Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis","volume":"828 ","pages":"Article 111848"},"PeriodicalIF":1.5000,"publicationDate":"2023-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Evaluation of the effects of simulated hypoxia by CoCl2 on radioresistance and change of hypoxia-inducible factors in human glioblastoma U87 tumor cell line\",\"authors\":\"Elham Khakshour , Mohammad Taghi Bahreyni-Toossi , Kazem Anvari , Mohammad Amin Shahram , Fereshteh Vaziri-Nezamdoust , Hosein Azimian\",\"doi\":\"10.1016/j.mrfmmm.2023.111848\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Purpose</h3><p>Glioblastoma<span><span> (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 </span>radioresistance<span> 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.</span></span></p></div><div><h3>Methods</h3><p>Following the treatment of cells with different concentrations of CoCl<sub>2</sub><span>, an MTT assay was used to evaluate the cytotoxicity of CoCl</span><sub>2</sub><span>. To understand the effects of Ionizing radiation on CoCl</span><sub>2</sub>-treated groups, cells were exposed to irradiation after pretreating with 100 μM CoCl<sub>2,</sub><span> 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<span> (DCFDA) probe staining. Additionally, the expression of hypoxia-associated genes, including HIF-1α, HIF-2α, and their target genes (GLUT-1), was monitored by reverse transcription polymerase chain reaction (RT-PCR).</span></span></p></div><div><h3>Results</h3><p><span>Our study revealed that the cell viability of CoCl</span><sub>2</sub>-treated cells was decreased in a concentration-dependent manner. Also, CoCl<sub>2</sub> did not cause any cytotoxicity on U87 cells at a concentration of 100 μM after treatment for 24 h. Colony formation assay showed that CoCl<sub>2</sub><span> pretreatment induced radioresistance of tumor cells compared to non-treated cells. Also, CoCl</span><sub>2</sub> can protect cells against irradiation by the clearance of ROS. Moreover, Real-time results showed that the mRNA expression of HIF-1α and GLUT-1 were significantly upregulated following hypoxia induction and/or irradiation condition. However, the level of HIF-2α mRNA did not change significantly in hypoxia or irradiation alone conditions, but it increased significantly only in hypoxia + irradiation conditions.</p></div><div><h3>Conclusion</h3><p>Taken together, our results indicated that simulating hypoxia by CoCl<sub>2</sub> can effectively increase hypoxia-associated genes, specially HIF-1α and GLUT-1, but did not affect HIF-2α gene expression. Also, it can increase the clearance of ROS, respectively, and it leads to inducing radioresistance of U87 cells.</p></div>\",\"PeriodicalId\":49790,\"journal\":{\"name\":\"Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis\",\"volume\":\"828 \",\"pages\":\"Article 111848\"},\"PeriodicalIF\":1.5000,\"publicationDate\":\"2023-12-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0027510723000350\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0027510723000350","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
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 μM 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-1α, HIF-2α, 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 concentration-dependent manner. Also, CoCl2 did not cause any cytotoxicity on U87 cells at a concentration of 100 μM 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-1α and GLUT-1 were significantly upregulated following hypoxia induction and/or irradiation condition. However, the level of HIF-2α 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-1α and GLUT-1, but did not affect HIF-2α gene expression. Also, it can increase the clearance of ROS, respectively, and it leads to inducing radioresistance of U87 cells.
期刊介绍：
Mutation Research (MR) provides a platform for publishing all aspects of DNA mutations and epimutations, from basic evolutionary aspects to translational applications in genetic and epigenetic diagnostics and therapy. Mutations are defined as all possible alterations in DNA sequence and sequence organization, from point mutations to genome structural variation, chromosomal aberrations and aneuploidy. Epimutations are defined as alterations in the epigenome, i.e., changes in DNA methylation, histone modification and small regulatory RNAs.
MR publishes articles in the following areas:
Of special interest are basic mechanisms through which DNA damage and mutations impact development and differentiation, stem cell biology and cell fate in general, including various forms of cell death and cellular senescence.
The study of genome instability in human molecular epidemiology and in relation to complex phenotypes, such as human disease, is considered a growing area of importance.
Mechanisms of (epi)mutation induction, for example, during DNA repair, replication or recombination; novel methods of (epi)mutation detection, with a focus on ultra-high-throughput sequencing.
Landscape of somatic mutations and epimutations in cancer and aging.
Role of de novo mutations in human disease and aging; mutations in population genomics.
Interactions between mutations and epimutations.
The role of epimutations in chromatin structure and function.
Mitochondrial DNA mutations and their consequences in terms of human disease and aging.
Novel ways to generate mutations and epimutations in cell lines and animal models.
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