Screening And Mechanism Of Antitumor Components Based On NF-?B Reporter Gene Model

Objective:Constructing a high throughput screening model of inhibitors based on reporter gene targeting NF-kappa B signaling pathway.The anti-tumor active ingredients based on NF-kappa B signaling pathway were screened,and the anti-tumor mechanism and combined administration of the active ingredients were discussed.Methods:1.Transfecting pNF-kappa B-luc reporter gene plasmid and Renal Luciferase plasmid from the sea cucumber into Hek293 cells,the effects of different dosage,time and concentration of administration on the construction of the reporter gene model were investigated,and a double luciferase reporter gene model was constructed.Using PDTC,an inhibitor of NF-kappa B,as a positive drug,the inhibitor of NF-kappa B pathway was screened based on LPS-induced activation.2.CCK-8 method was used to determine the cytotoxicity of cardanin A?RDA?and IC₅₀0 was calculated.Immunofluorescence assay was used to study the activation-nuclear transport of RDA on NF-kappa B p65 and NF-kappa B p50.RT-PCR was used to study the effect of RDA on the transcription of RNA in the NF-kappa B pathway.Western Blot was used to detect the changes of the expression of active proteins NF-kappa B p65,IkB?,MAPK p38 and JNK in the NF-kappa B pathway.3.CCK-8 assay was used to detect the inhibitory effect of combined administration of dicitabine and RDA on QGY-7703 hepatocellular carcinoma cells;Transwell experiment was used to study the effect of combined administration of RDA and cetabine on invasion and migration of QGY-7703 cells;Annexin V-FITC/PI double staining was used to detect the effect of combined administration of RDA and cetabine on apoptosis of tumor cells.Results:1.Fluorescence observation showed that pNF-kappa B-luc and green fluorescent protein particles were successfully co-transfected into Hek293T cells.High throughput screening results showed that the main component of the two tips,rhododendrin A?RDA?,was an inhibitor of NF-kappa B signaling pathway and had a good inhibitory effect on NF-kappa B signaling pathway.2.The half inhibitory concentration of RDA on QGY-7703 was 5.74 umol/L at 48 h.Immunofluorescence assay showed that RDA could effectively inhibit the nuclear transport of NF-kappa B P50 and NF-kappa B p65 in the results of DAPI staining.The results of nuclear staining showed that RDA could effectively inhibit the nuclear transport of NF-kappa B P50 and NF-kappa B p65.RT-PCR results showed that RDA had a good specific inhibitory effect on the NF-kappa B signaling pathway;Western-blot results showed that RDA could reduce the phosphorylation of NF-kappa B p65,IkB?,MAPK p38,JNK subunits,and significantly inhibit the NF-kappa B signaling pathway,so as to achieve the purpose of anti-cancer.3.The inhibition rates of migration and invasion of QGY-7703 cells were 81.16%and 84.48%respectively after RDA combined with ceftazidime,and the apoptotic rate of QGY-7703 cells was 67.7%after 48 hours of combined administration.Conclusion:In this paper,we successfully constructed an anti-tumor active component model based on the report gene of NF-kappa B.Bamboo charcoal A has anti-tumor effect.It can play an anti-tumor role by inhibiting the expression of NF-kappa B p65/p50.Combined administration can promote the apoptosis of cancer cells and increase their anti-tumor activity.This study laid a foundation for the anti-tumor mechanism of bamboo charcoal A.