DMA, a Bisbenzimidazole, Offers Radioprotection by Promoting NFκB Transactivation through NIK/IKK in Human Glioma Cells

BackgroundIonizing radiation (IR) exposure often occurs for human beings through occupational, medical, environmental, accidental and/or other sources. Thus, the role of radioprotector is essential to overcome the complex series of overlapping responses to radiation induced DNA damage.
Methods and ResultsTreatment of human glioma U87 cells with DMA (5- {4-methylpiperazin-1-yl}-2-[2′-(3, 4-dimethoxyphenyl)-5′-benzimidazolyl] in the presence or absence of radiation uncovered differential regulation of an array of genes and proteins using microarray and 2D PAGE techniques. Pathway construction followed by relative quantitation of gene expression of the identified proteins and their interacting partners led to the identification of MAP3K14 (NFκB inducing kinase, NIK) as the candidate gene affected in response to DMA. Subsequently, over expression and knock down of NIK suggested that DMA affects NFκB inducing kinase mediated phosphorylation of IKKα and IKKβ both alone and in the presence of ionizing radiation (IR). The TNF-α induced NFκB dependent luciferase reporter assay demonstrated 1.65, 2.26 and 3.62 fold increase in NFκB activation at 10, 25 and 50 µM DMA concentrations respectively, compared to control cells. This activation was further increased by 5.8 fold in drug + radiation (50 µM +8.5 Gy) treated cells in comparison to control. We observed 51% radioprotection in control siRNA transfected cells that attenuated to 15% in siRNA NIK treated U87 cells, irradiated in presence of DMA at 24 h.
ConclusionsOur studies show that NIK/IKK mediated NFκB activation is more intensified in cells over expressing NIK and treated with DMA, alone or in combination with ionizing radiation, indicating that DMA promotes NIK mediated NFκB signaling. This subsequently leads to the radioprotective effect exhibited by DMA.

背景 人类常通过职业暴露、医疗应用、环境接触、意外事故及其他途径接触电离辐射（Ionizing Radiation, IR）。因此，辐射防护剂在应对辐射诱导的DNA损伤所引发的一系列复杂重叠应答过程中，发挥着不可或缺的关键作用。
方法与结果 采用微阵列（microarray）与二维凝胶电泳（2D PAGE）技术，检测在有无电离辐射的条件下，使用DMA（5-{4-甲基哌嗪-1-基}-2-[2′-(3,4-二甲氧基苯基)-5′-苯并咪唑基]）处理人胶质瘤U87细胞后，多组基因与蛋白质的差异调控情况。通过构建信号通路并对已鉴定蛋白及其互作伴侣的基因表达进行相对定量分析，确定MAP3K14（核因子κB诱导激酶，NFκB inducing kinase, NIK）为受DMA调控的候选基因。后续通过NIK的过表达与敲低实验证实，无论是否存在电离辐射，DMA均可影响核因子κB诱导激酶介导的IKKα与IKKβ磷酸化过程。肿瘤坏死因子-α（TNF-α）诱导的核因子κB依赖性荧光素酶报告基因实验显示，相较于对照组细胞，在DMA浓度分别为10、25和50 μM时，NFκB激活水平分别升高1.65、2.26和3.62倍；在药物联合电离辐射（50 μM DMA+8.5 Gy）处理组中，该激活水平较对照组进一步提升5.8倍。实验观察到，转染对照siRNA的U87细胞在经DMA联合电离辐射处理24小时后，可实现51%的辐射防护效果，而在经NIK siRNA处理的细胞中，该防护效果减弱至15%。
结论 本研究表明，在过表达NIK并单独或联合电离辐射处理的细胞中，NIK/IKK介导的NFκB激活水平显著增强，提示DMA可促进NIK介导的NFκB信号通路，进而产生辐射防护效应。