Identification and application of anti-inflammatory compounds screening system based on RAW264.7 cells stably expressing NF-ΚB-dependent SEAP reporter gene

Abstract

Background: NF-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. Method: We 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. Result: In 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. Conclusion: In conclusion, RAW264.7 cells, stably expressing the NF-ΚB-dependent SEAP-reporter gene, may provide a new, feasible, and efficient cellular drug-screening system.