Abstract
Background: Gliomas possess low immunogenicity, which is an inevitable hinder in front of cancer immunotherapy. Different interferons (IFNs) may proceed apoptosis instead in p53-dependent or independent pathways. P53 induces the anti-inflammatory programmed cell death in cancer cells; on the other hand, IFN gamma (IFNγ) is a modulatory/pro-inflammatory cytokine. There are contradictory reports of whether this cytokine can possess an anti- or pro-cancerous impact on tumors. Hence, we aimed to investigate the possible cooperative apoptotic effect of the P53 and IFNγ over expressions on the U87 glioblastoma cell line. Materials and Methods: The P53 expressing vector was amplified by Escherichia coli BL21. This vector was confirmed by the aid of sequencing. At the next step, U87 cells were transfected using lipofectamine. Cells were treated with P53 vector and/or IFNγ. The type of cellular death investigated by flow cytometry and the expression level of cleaved caspase-3 protein was also precisely demonstrated by western blotting. Results: Sequencing results revealed that inserted P53 was identical with human P53. Western blot results revealed that both IFNγ and P53 overexpression could up-regulate cleaved caspase-3 protein expression in this cell line. Interestingly, flow cytometry data determined that concurrent treatment with P53 exogenous overexpression and IFNγ induces about 70% apoptosis in U87; more than the sum of cell death occurs after IFNγ or P53 overexpression alone (~18%+21%=39%). Conclusion: The present study results showed that p53-overexpression and IFNγ could ultimately induce up-regulation of the caspase-3 and ultimately significant apoptosis increasing in the U87 cell line. Although IFNγ is believed to be a pro-inflammatory cytokine and P53 is an anti-inflammatory agent, our results demonstrated that they could act synergistically to induce apoptosis in U87 cells. [GMJ.2021;10:e2270]
Highlights
Glioblastoma is the most aggressive and challenging type of brain tumor; it is a high-grade astrocytoma that can develop in adults and children [1]
Plasmid Extraction Competent E. coli BL21 transformed for amplification of P53 plasmid
Transfection Control To ensure that the transfection was performed functional, we utilized a eukaryotic GFP containing plasmid and transfected U87 cells with this plasmid as the same protocol used for the P53 plasmid
Summary
Glioblastoma is the most aggressive and challenging type of brain tumor; it is a high-grade astrocytoma that can develop in adults and children [1]. IFNγ is commonly secreted from natural immune cells, including T-cells found in the tumors’ microenvironment and the tumor cells at low concentrations [7] This master regulator can be beneficial in the immunotherapy of cancers with poor immunogenicity, as it is a pro-inflammatory cytokine and, according to the literature, upregulates major histocompatibility (MHC) class I [7]. Western blot results revealed that both IFNγ and P53 overexpression could up-regulate cleaved caspase-3 protein expression in this cell line. IFNγ is believed to be a pro-inflammatory cytokine and P53 is an anti-inflammatory agent, our results demonstrated that they could act synergistically to induce apoptosis in U87 cells
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