title: Identification and application of anti-inflammatory compounds screening system based on RAW264.7 cells stably expressing NF-κB-dependent SEAP reporter gene.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Reproductive Medicine, The Fourth Hospital of Xi'an, Xi'an, Shaanxi, People's Republic of China.
authors.forename: Juan
authors.initials: J
authors.lastname: Ren
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Xi
authors.initials: X
authors.lastname: Lan
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yi
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Sanqi
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China. lidongm@mail.xjtu.edu.cn.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi, 710061, People's Republic of China. lidongm@mail.xjtu.edu.cn.
authors.forename: Dongmin
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, People's Republic of China. lushemin@mail.xjtu.edu.cn.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi, 710061, People's Republic of China. lushemin@mail.xjtu.edu.cn.
authors.forename: Shemin
authors.initials: S
authors.lastname: Lu
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015870:Gene Expression; D017930:Genes, Reporter / Q000187:drug effects* / Q000502:physiology; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D016328:NF-kappa B / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000893:Anti-Inflammatory Agents; D016328:NF-kappa B; D000469:Alkaline Phosphatase
keywords: Drug screening; Inflammation; Reporter gene; Signal pathway