Construction Of High Throughput Screening System Based On NF-κB Signaling Pathway

NF-κB, a ubiquitous eukaryotic transcription factor in mammals, activates the transcription and subsequent expression of more than 150 genes implicated in apoptosis, cell adhesion, proliferation, the innate- and adaptive-immune responses, inflammation, cellular stress response and tissue remodeling. Constitutive activation of NF-κB was documented in inflammatory diseases such as rheumatoid arthritis, atherosclerosis, multiple sclerosis and asthma and several cancers such as prostate cancer, gliomas and lymphomas. Inhibition of activation of NF-κB was reportedly attributable to the treatment of these NF-κB-involved diseases, so NF-κB has became a pivotal target in the treatment of inflammatory diseases and cancers, physiological processes during which an aberrant activation of NF-κB occurs. In this work, we constructed high throughput screening system targeted to NF-κB. According to the knowledge accumulated that there is an universal NF-κB response element sequence in target genes of NF-κB, four tandem repeats for NF-κB binding were constructed upstream of minimal promoter of CMV, and then the two elements were cloned into multiple cloning site of pGL4.20 from Promega Corporation. With a NF-κB responsive luciferase reporter gene integrated, the plasmid pGL4.20-κB-miniCMV was constructed, ensuring the transfection of pGL4.20-κB-miniCMV into human embryonic kidney cells 293T and human prostate cancer cells DU145 by lipofectamine 2000, respectively. For selection, puromycin was added as stress to select the stable cell lines in which the plasmids were integrated into the genomes. Upon validation, one clone from 293T cells, numbered 293T-κB-2, responded to NF-κB stimuli well and the other clone from DU145 cells numbered DU145-κB-5 responded to PDTC significantly, which inhibits the activation of NF-κB. Using the cell line constructed in this work, a natural products library purchased from National Compound Resource Center was screened, yielding 33 compounds capable of inhibiting NF-κB activation by approx. 45%. In a word, high throughput screening system based on NF-κB was constructed and was applied to screen inhibitors of NF-κB successfully.