Effect And Mechanism Of Bifendatatum Combined With Gefitinib On Lung Adenocarcinoma PC9 Cells And Balb/c Tumor-bearing Nude Mice

Objective: To explore the combined effects of bifendatatum and gefitinib in lung adenocarcinoma PC9 cells and Balb/c tumor-bearing nude mice and explore its possible mechanism in PC9 cells,then explore the traditional Chinese medicine intervention method for therapy of lung adenocarcinoma.Methods: MTT,Soft Agar and Invasion were used to detect the viability and inhibitory effects of proliferation and invasion of PC9 cells by DMSO,BFD,gefitinib and BFD combined with gefitinib.Detected the expression levels of EGFR and downstream pAKT by Western blot.The lung adenocarcinoma PC9 cells were inoculated subcutaneously into the back of the neck of Balb/c nude mice,the mice were intragastrically administered with BFD,gefitinib,BFD combined with gefitinib,and the control group was intragastrically administered with 0.5%CMC.Evaluated the efficacy of BFD,gefitinib and combined intervention of the two drugs by tumor formation size,tumor weight,tumor inhibition rate,nude mice growth curve,and tumor volume growth curve.Judged the effect of the combination of two drugs by Jin Zhengjun method.Meanwhile,analyzed the transcription factor CTCF and expression levels of EGFR and their possible correlation in lung adenocarcinoma tissues by database.Transfected PC9 cells by HA-CTCF vector,ChIP was used to detect the binding and relationship between transcription factor CTCF and EGFR,proved that BFD regulates EGFR through CTCF.Western blot and PCR confirmed the expression of BFD on EGFR and downstream pAKT by CTCF.And detected the regulation of BFD on CTCF in PC9 cells.Soft Agar and Invasion were used to detect the regulation of BFD and CTCF on proliferation and invasion of PC9 cells.Results: Compared with gefitinib,BFD combined with gefitinib significantly inhibited the proliferation,colony and invasion of lung adenocarcinoma PC9 cells.At the same time,the expression level of pAKT in the downstream of EGFR signaling pathway could be down-regulated(p<0.01).In the subcutaneous tumor formation experiment of nude mice,compared with gefitinib,gefitinib combined with BFD significantly reduced the tumor size,the tumor weight,and slowed down the growth of the tumor(p<0.05),and the two drugs had synergistic effect.In the analysis database,there were four sites in the nucleotide sequence of the EGFR gene promoter region that could be combined with a common binding site on the transcription factor CTCF.While high expression of transcription factor CTCF and EGFR was a risk factor for lung adenocarcinoma.In the cell lines PC9 transfected with HA-CTCF vector,the expression level of EGFR was significantly increased.After BFD intervention,the expression level of CTCF was significantly decreased,further reducing the expression level of EGFR,and reducing cell proliferation,invasion,migration(p<0.01).Conclusion: BFD can enhance the inhibition of gefitinib on the function of lung adenocarcinoma PC9 cells and the anti-tumor effect of Balb/c tumor-bearing nude mice.The transcription factor CTCF can bind the promoter region of EGFR,and promote the expression level of EGFR in PC9 cells.BFD can reduce the expression level of EGFR and pAKT,a key factor in downstream signaling pathway by transcription factor CTCF,which has combined effect with gefitinib in PC9 cells.