The Establishment And Application Of Cell Models For Antitumor Drug Screening Among Anthraquinone Compounds Based On The Target Of Egfr By Luciferase Reporter Gene System

Objectives1.The purpose of this study is to establish luciferase reporter gene expression cell models of A549-EGFR-Luc2 and CNE1-EGFR-Luc2 cells based on the target of EGFR promoter.2.Luciferase reporter system is used to screen active anti-tumor candidate compounds targeted inhibiting EGFR at transcriptional level.3.It is designed to reveal the relationship of targeted regulation of EGFR with its anti-tumor activities and provide new strategies for the development and discovery of novel antitumor agents.Methods1.The expressions of EGFR in different types of cells including nasopharyngeal carcinoma cells,breast carcinoma cells,cervical cancer cells,Lung carcinoma cells,together with normal human nasopharyngeal epithelial NP69 cells and normal epithelial mammary MCF-10 A cells were determined by western blot analysis.According to the results,the cells would be selected as the experimental models if EGFR was over-expressed.2.Using genetic recombination technology,a lentiviral carrying luciferase reporter vector recombined with EGFR promoter was successfully constructed,which was called LV-EGFRpromoter-Luc2.Both A549 and CNE1 cells with high expression of EGFR were infected with EGFR-promoter-Luc2 lentiviral to obtained cell lines that stably expressed firefly luciferase,named A549-EGFR-Luc2 and CNE1-EGFR-Luc2 cells,respectively;3.The important conditions of the luciferase reporter assay of drug screening system were optimized and controlled.In addition,luciferase reporter assay was used to screen Gefinitib,Cisplatin,lead compound Rhein and anthraquinonoid compound 4a,B,C,H,I,J,K and N that targeted regulating the promoter activities of EGFR in A549-EGFR-Luc2 and CNE1-EGFR-Luc2 cells.4.MTT assay was applied to detect the half inhibitory concentrations(IC50)of Gefinitib,Cisplatin,lead compound Rhein,anthraquinonoid compound 4a,B,C,H,I,J,K and N on A549 and CNE1 cells.And the effects of different compounds on EGFR expression in A549 and CNE1 cells were detected by Western Blot analysis.5.A549 and CNE1 cells were selected as the experimental models to test the effects of Gefinitib,Cisplatin,lead compound Rhein and anthraquinonoid compounds 4a,B,C,H combined with radiotherapy on the following indicators of the two cell lines.(1)MTT assay was used to detected inhibition rates of cells treated with different compounds combined with or without irradiation.(2)The colony-forming assay was used to detect the radiosensitization activities of the candidate compounds on A549 and CNE1 cells.(3)The expressions of EGFR in different treatments groups were detected by western blot analysis.(4)Comet assay was used to detect the DNA damage of the cells after dealt with different treatments.6.Immunofluorescence assay was applied to explore the effects of anthraquinonoid compounds 4a,H and chemotherapeutic drug paclitaxel on formation of F-actin filaments.Results1.Western Blot analysis results suggest that A549 and CNE1 cells possess high expression level of EGFR among different types of test cell lines.2.According to the identification results by complete genome sequencing and double enzyme digestion,a lentiviral expression vector carrying luciferase reporter vector recombined with EGFR promoter has been successfully constructed.Both A549 and CNE1 cells are infected with the lentivirus and screened by puromycin to obtain A549-EGFR-Luc2 and CNE1-EGFR-Luc2 cells that stably expressing firefly luciferase.Besides,luciferase reporter assay shows that fold expression of the luciferase activities in A549-EGFR-Luc2 and CNE1-EGFR-Luc2 cells are 49 and 43 times of the negative control,respectively.3.From the results of the optimal experiment of luciferase reporter gene drug screening system,both luciferase activity and the expression of luciferase are on the high level when 5000 cells per well are seeded,reaction temperature controls at 25℃ and cell lysis time is 5 minutes.Therefore,it is chosen as the immobilized condition for the further drug screening system based on luciferase reporter assays.Luciferase reporter assays suggest that Gefinitib together with anthraquinonoid compounds 4a,B,C,H show good inhibition on EGFR promoter in both A549-EGFR-Luc2 and CNE1-EGFR-Luc2 cells.4.MTT results suggest that Gefinitib exhibits good inhibition activities on A549 and CNE1 cells.And besides,the inhibition effects of the anthraquinonoid compounds 4a,B,C,H,I and N on CNE1 cells are much stronger than that on A549 cells.The anti-tumor activities of compounds H and N on A549 and CNE1 cells are significantly increasing compared with the lead compound Rhein(P<0.001).5.Western Blotting analysis results reveal that Gefinitib,compound 4a,B,C and H show more prominent activities than the lead compound Rhein in down-regulating the protein expression of EGFR through targeted inhibiting the transcription activity of EGFR promoter in both A549 and CNE1 cells.6.Cells are dealt with different compounds combined with radiotherapy before detecting the following indicators.(1)MTT results show that the inhibition rates of the cells are relatively low in the groups treated with compounds alone and irradiation alone,while the combined treatment greatly increase the inhibition rates of A549 and CNE1 cells.Among which,the combined application of radiotherapy and anthraquinonoid compounds 4a,B,C and H significantly increase the inhibition of the cells compared with the group treated with irradiation alone(P<0.001).(2)The colony forming assay suggests that Gefinitib,anthraquinonoid compounds 4a,C and H show significant radiosensitization effects on A549 cells compared with Rhein,the SER of which are 1.462,1.619,1.708 and 1.290,respectively.As to CNE1 cells,Cisplatin,4a and C show remarkable radiosensitization activities which is superior to the lead compound Rhein,the corresponding SER are 1.256,1.184 and 1.206,respectively.(3)According to Western Blot analysis results,radiotherapy obviously activates the expression of EGFR,while the application of Gefinitib,anthraquinonoid compounds 4a,B,C and H significantly down-regulate the expression of EGFR in both A549 and CNE1 cells.(4)Radiotherapy alone shows little effects on DNA damage of the cells,while the combined application of Gefinitib,anthraquinonoid compounds 4a,C and H significantly increase the DNA fragmentations,and the DNA percentage in tail are higher than that of irradiation alone group and the groups treated with anthraquinonoid compounds alone(P<0.05).7.Immunofluorescent assay results show that the expressions of F-actin microfilament in A549 and CNE1 cells are significantly decreasing,the microfilament cytoskeletons are rearranging,followed by the appearance of the typical F-actin rings at nuclear of the cells,thereby resulting in the loss of cytoskeleton integrity in both A549 and CNE1 cells after treated with anthraquinonoid compounds 4a and H with the extension of time.Conclusions1.Luciferase reporter system based on EGFR promoter has been successfully established and the cell models for screening active antitumor compounds targeting inhibition EGFR at transcriptional level are constructed followed.2.The antitumor activities and targeting inhibitory activities on EGFR transcription of anthraquinonoid compounds 4a,B,C and H are superior to the lead compound Rhein.3.Anthraquinonoid compounds 4a,C and H without cytotoxic exhibit good radiosensitizing effects on Lung carcinoma A549 cells and nasopharyngeal carcinoma CNE1 cells when combined with radiotherapy which is superior to lead compound Rhein.4.Anthraquinonoid compounds 4a and H promote depolymerization and rearrangement of F-actin filaments,inducing destruction of microfilament skeleton system,thereby resulting in apoptosis in tumor cells through inhibiting expression of EGFR and blocking the activation of its downstream passway.