Mechanism Of BPI-15086 On EGFR-T790M In Vitro

Objective:To investigate the effects of BPI-15086 on human lung proliferation and the expression of EMT-related protein E-cadherind in adenocarcinoma cell lines PC9 and A549 carrying EGFR wild-type human lung adenocarcinoma,exon-sensitive mutation of EGFR 19,the role and cell cycle of human adenocarcinoma cell line H1975 carrying EGFR T790M mutation and the effect of apoptosis.Methods:The mutation types of EGFR gene in three cell lines were identified by real-time PCR with ARMS;the inhibition rates of proliferation of A549,PC9 and H1975 cells were detected by CCK8 method;the apoptosis of H-1975,PC9 and A549 cells was detected by flow cytometry AnnexinV method after 24 hours of treatment with blank group and specific concentration of BPI-15086;and the apoptosis of H-1975,PC9 and A549 cells was detected by flow cytometry.The cell cycle of 549 cells was changed 24 hours after treatment,and the expression of EMT-related E-cad was detected by Western-blot at different concentrations of BPI-15086.Results:1.CCK8 results showed that BPI-15086 could inhibit the proliferation of A549,PC9 and H1975 cells in a concentration-dependent manner(P<0.01).The inhibitory effect of BPI-15086 on the proliferation of H1975 cells was significant(P<0.05).2.24 hours after BPI-15086 acted on A549,PC9 and H1975 cells,the morphological changes of cells were observed under electron microscopy:the cell volume was reduced,the junction disappeared,the cells detached from the surrounding cells,the density of cytoplasm increased,and the nuclear plasm concentrated.AnnexinV-FITC/PI staining showed that BPI-15086 could induce apoptosis of A549,PC9 and H1975 cells(P<0.05).3.24 hours after BPI-15086 acted on A549,PC9 and H1975 cells,flow cytometry showed that A549,PC9 and H1975 cells had G1/G0 arrest(P<0.05),and S phase decreased(P<0.05).4.24 hours after BPI-15086 acted on A549,PC9 and H1975 cells,Western blot results showed that E-cadherin protein expression increased.CONCLUSION:The third generation of EGFR-TKI drug BPI-15086 can inhibit the proliferation of A549,PC9 and H1975 cells and promote their apoptosis.The effects of inhibiting proliferation and promoting apoptosis were obvious in H1975 cells,suggesting that drug-resistant H1975 cells carrying EGFRT790M/L858R mutation were highly sensitive to BPI-15086.When BPI-15086 acts on A549,PC9 and H1975 cells,G1/G0 blockade and S phase ratio decrease,which may inhibit the DNA replication of tumor cells and reduce the activity of tumor cells,so as to control tumors.BPI-15086 may reverse epithelial interstitialization and reduce the invasiveness of tumors by up-regulating the expression of E-cadherin protein.