The Study Of The Mechanism Of Yiqi Chutan Tang In Intervention Of EGFR-TKIs Acquired Drug Resistance In Lung Cancer

ObjectiveIn this study,we will explore the anti-cancer effect of Yiqi Chutan Tang(YFSJ)on gefitinib-resistant human non-small cell lung cancer(NSCLC)cell lines H1975 and H1650,and to identify potential molecular mechanisms associated with inhibition of acquired resistance.MethodsThe first part:(1)XTT assay was used to detect the effects of YFSJ or gefitinib on cell viability in human NSCLC cell lines H1975 and H1650.(2)The effects of YFSJ and gefitinib on the proliferation of human EGFR-mutant NSCLC cell lines H1975 was detected by EdU assay.(3)Cell cycle assay were performed by flow cytometry to investigate the effects of YFSJ,gefitinib and YFSJ combined with gefitinib on the cell cycle of human EGFR-mutant NSCLC cell lines.(4)Flow cytometry was used to detect PI3K,Akt,mTOR,LC3A and LC3B protein experssion in the PI3K/Akt/mTOR pathway and autophagy in NSCLC cell lines H1975 and H1650 with the treatment of YFSJ and YFSJ combined withgefitinib.(5)The mTOR inhibitor and autophagy activator was used to identify the effects and mechanism of YFSJ.The second part:(1)Transcriptome sequencing was used to detect the effects of YFSJ on differential gene expression in NSCLC cells.(2)Gene Ontology and Kyoto Encyclopedia Gene and Genomics enrichment analysis were used to explore the effects of YFSJ on the molecular mechanism in NSCLC cells.(3)Xenograft nude mouse model of lung cancer was used in vivo to detect the effects of YFSJ in inhibiting cancer and reducing acquired drug resistance,as well as exploring its underlying mechanism.The third part:(1)The xenograft model of nude mice was used toinvestigate the effects of YFSJ on mice weight,tumor volume and tumor weight.(2)HE staining assay was used to explore the effects of YFSJ on the tumor morphology of nude mice.(3)Western blot assay was used to explore the effects of YFSJ on PI3K/Akt/mTOR pathway and autophagy.ResultsThe first part:(1)XTT cell viability assay showed that YFSJ inhibited the cell viability of H1975 and H1650 in a dose-dependent fashion.YFSJ combined with gefitinib significantly reduced the cell viability of H1975 and H1650 cells in a dose-dependent fashion at 24 and 48 hours.These results indicated that YFSJ could reduce the resistance to gefitinib in NSCLC resistant cell lines.(2)EdU assay showed that gefitinib alone could inhibit cell proliferation,although the use of YFSJ could not inhibit cell proliferation,YFSJ could increase the inhibition effects of gefitinib on the proliferation in H1975 cells in a dose-dependent fashion.(3)Flow cytometry assay showed that the YFSJ combined with gefitinib and gefitinib alone could significantly arrest H1975 and H1650 cells in G0/G1 phase.(4)Flow cytometry assay showed that the expression of PI3K,Akt and mTOR were down-regulated,while the expression of LC3A and LC3B were up-regulated in both H1975 and H1650 cells after 24 hours and 48 hours after YFSJ and YFSJ combined with gefitinib treatment,which indicated that PI3K/Akt/mTOR pathway and autophagy were important targets in EGFR-TKIs resistant NSCLC cell lines of YFSJ combined with gefitinib treatment.(5)YFSJ might be an effective mTOR inhibitor and autophagy activator,which could reverse the gefitinib resistance by down-regulating the PI3K/Akt/mTOR pathway and up-regulating autophagy.The second part:(1)Transcriptome sequencing showed that the differential gene expression in H1975 cells after YFSJ combined with gefitinib treatment was very different from YFSJ or gefitinib alone,it might be the reason of different molecular mechanisms regulation.(2)In the GO functional enrichment analysis,more up-regulated genes were observed in the treatment of two different doses of YFSJ,while more down-regulated genes were observed in gefitinib group.Similar results were also showed in the KEGG pathway analysis.These results indicated that the regulation of YFSJ and gefitinib on the genetic level in H1975 cells was different.In GO and KEGG analysis,cell cycle and its related terms were observed,which indicated that the cell cycle was significantly regulated at the genetic level.Lung cancer,other cancers and cancer-related pathways were observed,indicating that the inhibition effects of YFSJ on cancer might related to its ability to regulate multiple related pathways of cancer.(3)In the EFGR-TKIs resistance pathway and NSCLC pathway,high and low doses of YFSJ,gefitinib,high and low doses of YFSJ combined with gefitinib affected the PI3K-Akt Signal pathway.These indicated that the PI3K/Akt pathway was a target in EGFR-TKIs resistant NSCLC cell lines.The third part:(1)YFSJ could effectively inhibit the tumor volume and tumor weight in vivo,which indicated YFSJ could reduce the resistance to gefitinib in xenograft model of nude mice.(2)The results of HE staining assay showed that YFSJ and gefitinib could increase tumor necrosis in xenograft model of nude mice;the combined group resulted in the highest tumor necrosis in nude mice,indicating the significant effects of combined group.(3)Western blot assay showed that YFSJ and YFSJ combined with gefitinb could inhibit the expression of PI3K,Akt and mTOR,increase LC3A and LC3B in vivo,which indicated that YFSJ can inhibit the PI3K/Akt/mTOR pathway and increase the expression of autophagy.ConclusionYFSJ inhibited the cell viability of EGFR-TKIs resistance cell lines,reduced the resistance to gefitinib,arrested cell cycle and inhibited the tumor volume of xenograft nude mice.This might be the reason of down-regulating PI3K/Akt/mTOR pathway and up-regulating the autophagy.