Abstract
BackgroundNF-κB is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is therefore regarded as an important target for anti-inflammatory drug screening.MethodWe recombined the reporter gene vector with inserting the “neo” transcript into the vector pNF-κB-SEAP, made the reporter gene vector stable in a eukaryotic cell line. The recombinant reporter gene vector we named pNF-κB-SEAP-Neo was transfected into RAW264.7. We selected the transfected RAW264.7 cell line with G418 for 15 days and then get RAW264.7 cells stably expressing NF-κB-dependent SEAP named as RAW264.7-pNF-κB-SEAP cells. We treated the RAW264.7-pNF-κB-SEAP cells with NF-κB agonists as LPS, PolyI:C and TNF-α, NF-κB inhibitor as PDTC and BAY117085, in different concentrations and time points and tested the expression of the SEAP, constructed the drug screening system on the base of the RAW264.7-pNF-κB-SEAP cell line. 130 chemicals were screened with the drug screening system we constructed and one of these chemicals numbered w10 was found could inhibit the NF-κB significantly. At last, we verified the inhibition of w10 to expression of genes promoted with NF-κB in HepG2 and Hela, and to migration of Hela.ResultIn this study, we established a drug screening system based on RAW264.7 cells that stably expressed the NF-κB-dependent, SEAP reporter gene. To develop a standard method for drug screening using this reporter-gene cell line, the test approach of SEAP was optimized and basic conditions for drug screening were chosen. This included the initial cell number inoculated in a 96-well plate, the optimum agonist, inhibitor of NF-κB pathway and their concentrations during screening. Subsequently, 130 newly synthesized compounds were screened using the stable reporter-gene cell line. The anti-inflammatory effects of the candidate compounds obtained were further verified in 2 cancer cell lines. The results indicated that compound W10 (methyl 4-(4-(prop-2-yn-1-ylcarbamoyl) phenylcarbamoyl) benzoate) significantly inhibited SEAP production under the screening conditions. Further results confirmed that the precursor compound significantly inhibited the transcription of NF-κB target genes.ConclusionIn conclusion, RAW264.7 cells, stably expressing the NF-κB-dependent SEAP-reporter gene, may provide a new, feasible, and efficient cellular drug-screening system.
Highlights
nuclear factor-κB (NF-κB) is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is regarded as an important target for anti-inflammatory drug screening
LPS for 24 h menifested as column chart of relative activity of secreted alkaline phosphatase (SEAP) and dose-response stimulation curve with common logarithm of LPS concentration as the ordinate and the stimulation ratio (%) as the abscissa, R2 = 0.9643. d The RAW264.7-pNF-κB-SEAP cells were treated with PDTC at concentrations of 0.1, 1, 10, and 100 μM or 1‰ DMSO (v/v) for 6 h and treated with LPS in 100 ng/mL for 24 h
The effect of PDTC on RAW264.7-pNF-κB-SEAP cells was manifested as column chart of relative activity of SEAP and the dose-response inhibition curve between the log10 concentrations of PDTC and the inhibition ratio calculated from SEAP relative activity, R2 = 0.9802, their relation according to the common logarithmic equation. e The RAW264.7-pNF-κB-SEAP cells were treated with BAY117085 at concentrations of 0.1, 1, 10, and 50 μM or 1‰ DMSO (v/v) for 6 h and treated with LPS in 100 ng/mL for 24 h
Summary
NF-κB is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is regarded as an important target for anti-inflammatory drug screening. When acute inflammatory response is triggered, it lasts for a short period and is regulated by negative feedback signals Dysregulation of this feedback mechanism results in chronic inflammation, which is believed to be a key pathophysiological mechanism in various health disorders. In insulin resistance associated with metabolic syndrome, macrophages secrete proinflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-1β [4] while in primary and metastatic tumours, they provide a proinflammatory microenvironment for cancer cell growth [5]. Based on their role in inflammation, macrophage activation has been recognized as a target for anti-inflammation therapy. One of the signal transduction pathways involved during macrophage activation is the nuclear factor-κB (NF-κB) pathway
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