Study On The Mechanism Of Feiyiliu Mixture Delaying EGFR-TKI Drug Resistance In Non-Small Cell Lung Cancer

Objective:To observe the effect of Feiyiliu Mixture（FYLM）on improving the response rate of EGFR-TKI therapy,and to explore how FYLM combined with osimertinib delays the resistance to EGFR-TKI in EGFR mutant cell lines and EGFR mutant Lewis lung cancer xenograft mice through in vitro cell experiments and in vivo animal experiments.The molecular mechanism of drugs,in order to provide experimental support for the synergistic and attenuating effects of traditional Chinese medicine on targeted drugs.Methods:Part of in vitro cell experiments1.The main components of the FYLM-containing serum were analyzed using a combination of ultra-high performance liquid chromatography and quadrupole-orbitrap mass spectrometry（UPLC-Q-Orbitrap-MS）.2.Construct BaF3-EGFR^{Del19/T790M/C797S} cells as a cell model of osimertinib resistance by retrovirus infection.And Western blot method was used to identify the overexpression level of EGFR^{Del19/T790M/C797S} in BaF3-EGFR^{Del19/T790M/C797S}kinase cells.3.The effects of different concentrations of osimertinib or FYLM-containing serum on the viability of BaF3-EGFR^{Del19/T790M/C797S} kinase cells were detected by MTT method,and the optimal concentration of FYLM-containing serum intervening cells was determined,which was used as a low dose drug concentration for subsequent cell experiments.At the same time,the effect of FYLM-containing serum on the viability of H1975 cells was detected.Moreover,the effect of FYLM-containing serum combined with osimertinib on the proliferation of BaF3-EGFR^{Del19/T790M/C797S} kinase cells was detected by MTT method.4.Detection of cyclin B1（cyclin B1）,apoptosis-related proteins（Bcl-2 and cleaved caspase-3）,PRC1 protein,Wnt pathway-related proteins（β-catenin,c-Myc,c-Jun）and EGFR pathway-related proteins（p-EGFR,EGFR,Akt,p-Akt）expression levels in BaF3-EGFR^{Del19/T790M/C797S} kinase cells by FYLM-containing serum combined with osimertinib by Western blot,Part of in vivo animal experiments1.Infect mouse Lewis lung cancer cells（LLC）with lentiviral vectors carrying EGFR^{Del19/T790M/C797S} overexpressed genes and negative control gene lentiviral vectors by lentiviral infection,A cell line with long-term stable expression of EGFR^{Del19/T790M/C797S} mutation and negative control gene was constructed.The overexpression level of EGFR^{Del19/T790M/C797S} in LLC-EGFR^{Del19/T790M/C797S} kinase cells was identified by fluorescence intensity,q RT-PCR and Western blot.2.By planting the negative control gene or the LLC cell line stably expressing the EGFR^{Del19/T790M/C797S} mutation in C57BL/6J mice subcutaneously,to create an empty vector Lewis lung cancer mouse xenograft model and EGFR^{Del19/T790M/C797S} mutant Lewis lung cancer mouse xenograft model,as an animal model of osimertinib resistance.The changes of body weight and transplanted tumor of the two groups of mice were observed,and the pathological changes of the mice of the two groups were observed by H-E staining.3.The main components of FYLM were analyzed by UPLC-Q-Orbitrap-MS.4.After 2 weeks of intragastric administration of EGFR^{Del19/T790M/C797S}mutant Lewis lung cancer mouse model with FYLM combined with osimertinib,the body weight and changes of transplanted tumors in each group were observed,and the pathological morphology of mice in each group was observed by H-E staining Variety.In order to detect the safety of the combination of FYLM and osimertinib,the changes of liver and kidney function in mice were observed.5.The effect of FYLM combined with osimertinib on the expression of ki67 andβ-catenin protein in the tumor tissue of EGFR^{Del19/T790M/C797S} mutant Lewis lung cancer mouse xenograft model was detected by immunohistochemical method,and the apoptosis of mice in each group were detected by TUNEL staining.6.q RT-PCR method was used to detect the effect of FYLM combined with osimertinib on the expression of miR-1-3p in EGFR^{Del19/T790M/C797S} mutant Lewis lung cancer mouse xenograft tumor model.7.Western blot was used to detect the effects of FYLM combined with osimertinib on cyclin B1（cyclin B1）,apoptosis-related proteins（Bcl-2and cleaved caspase-3）,PRC1 protein,Wnt pathway-related proteins（β-catenin,c-Myc,c-Jun）and EGFR pathway-related proteins（p-EGFR,EGFR,Akt,p-Akt）expression levels in EGFR^{Del19/T790M/C797S} mutant Lewis lung cancer mouse xenograft model.Results:Part of in vitro cell experiments1.The EGFR protein level in BaF3-EGFR^{Del19/T790M/C797S} cells was significantly higher than that in BaF3 parental cells,indicating that the BaF3 kinase cell model stably expressing EGFR^{Del19/T790M/C797S} mutations was successfully constructed.When the concentration of osimertinib reached1108 nmol/L,it could inhibit the growth of BaF3-EGFR^{Del19/T790M/C797S} cells by half.It suggested that BaF3-EGFR^{Del19/T790M/C797S} cells were resistant to osimertinib.However,FYLM-containing serum can reduce the survival rate of BaF3-EGFR^{Del19/T790M/C797S} cells,and can inhibit the activity of H1975-EGFR^L858R/T790Mcells.2.FYLM-containing serum combined with osimertinib inhibited the proliferation of BaF3-EGFR^{Del19/T790M/C797S} cells,down-regulated the protein expression levels of cyclin B1 and Bcl-2,and up-regulated the protein expression of cleaved caspase-3.3.FYLM-containing serum combined with osimertinib inhibited the protein expression of PRC1,β-catenin,c-Myc,c-Jun,p-EGFR and p-Akt in BaF3-EGFR^{Del19/T790M/C797S} cells.And the inhibitory effect of the high dose group was more significant,but the difference was not statistically significant.Part of in vivo animal experiments1.After lentivirus infection,the green fluorescence intensities of empty vector-LLC cells and EGFR^{Del19/T790M/C797S}-LLC cells were strong.The EGFR gene and protein levels in EGFR^{Del19/T790M/C797S}-LLC cells were significantly higher than those in empty vector-LLC cells.It shows that the LLC cell model stably expressing the EGFR^{Del19/T790M/C797S} mutation is successful.2.EGFR^{Del19/T790M/C797S} mutant Lewis lung cancer mice transplanted tumor model group had heavier tumors than the empty vector group mice,and the tumor growth rate was faster,indicating that compared with the negative control gene,the overexpression of EGFR^{Del19/T790M/C797S} may cause the increased proliferation of LLC cells and further caused the faster growth of tumor tissue in mice.3.FYLM combined with osimertinib can inhibit tumor progression,significantly reduce the tumor weight of EGFRDel19/T790M/C797S mutant Lewis lung cancer mouse xenograft tumor model,reduce the nuclear density of tumor tissue,lighten the nuclear staining,and reduce the ratio of nucleus to cytoplasm.And there was no significant change in body weight,liver and kidney function of mice in each group,indicating that the combined use of FYLM and osimertinib has no significant toxic effect.4.FYLM combined with osimertinib inhibited the protein expression levels of ki67,cyclin B1,and Bcl-2 in tumor tissue of EGFR^{Del19/T790M/C797S} mutant Lewis lung cancer mouse xenograft model,while up-regulating the protein expression of cleaved caspase-3.5.FYLM combined with osimertinib elevates the expression of miR-1-3p in n tumor tissue of EGFR^{Del19/T790M/C797S}mutant Lewis lung cancer xenograft mouse model.6.FYLM combined with osimertinib inhibited the protein expression of PRC1,β-catenin,c-Myc,c-Jun,p-EGFR and p-Akt in tumor tissue of EGFR^{Del19/T790M/C797S} mutant Lewis lung cancer mouse xenograft model,and high-dose Group inhibition was more pronounced.Conclusion:1.The mutation of EGFR^{Del19/T790M/C797S} leads to the resistance of cells to osimertinib and accelerates the growth of transplanted tumors in tumor-bearing mice.2.FYLM combined with osimertinib reduces the proliferation and promotes apoptosis in EGFR^{Del19/T790M/C797S} mutant cells.3.FYLM combined with osimertinib can not only inhibit EGFR-triple mutation（Del19/T790M/C797S）cells,but also inhibit EGFR-double mutation（L858R/T790M）cells.Due to tumor heterogeneity,drug-resistant EGFR-mutant NSCLC patients carry more than one EGFR mutation,suggesting that the clinical application of FYLM can cover more drug-resistant mutant NSCLC patients.4.FYLM combined with osimertinib inhibited the growth of transplanted tumors in EGFR^{Del19/T790M/C797S} mutant Lewis lung cancer mice,reduced proliferation,and promoted apoptosis at the same time.5.FYLM combined with osimertinib may inhibit the PRC1/Wnt/EGFR pathway by activating miR-1-3p,thereby inhibiting the proliferation of drug-resistant mutant cells and promoting the apoptosis of drug-resistant mutant cells.6.FYLM combined with osimertinib is safe and effective.