Study On Inhibiting Human Non-small Cell Lung Cancer Cell Lines And Its Targets Of Aqueous Extract Of Taxus Chinensis Var.mairei(AETC)in Combination With Gefitinib

Objective To study the proliferative effects of Aqueous Extract of Taxus chinensis var.mairei(ATEC)in combination with gefitinib on human non-small cell lung cancer cell lines as well as their effects on ERK1/2、STAT3 signaling pathway.Clear the mechanism of ATEC overcoming Geditinib resistance.Methods Extracted from Taxus chinensis var cooking with water;we vitro cultured human lung adenocarcinoma cell lines PC9/R,H1975,A549,H820,PC9,.Each cell line was divided to blank contorl group,ATEC group,gefitinib group and AETC combined gefitinib group.After drugs effected the cells,measured the inhibit of cell proliferation by MTT,we tested each group’s optical density(OD)value and calulated the relative cell proliferation inhibition rate and combination index of 2 drugs.We used Real-time PCR to test the expression of EGFR,ERK1/2,STAT3 mRNA,Western-blot to test the expression of EGFR,p-EGFR,ERK1/2、STAT3,p-ERK1/2,p-STAT3.All the statistical analysis were performed by SPSS 19.0 software.Statistical significance was compared between each treatment groups and control group by one-way ANOVA.Values with P<0.05 were considered as statistically significant.Result 1.MTT method test:The aqueous extract of T.chinensis and gefitinib have inhibitory effect on the proliferation of human lung cancer cell A549,H1975,H820,PC9 and PC9/R,the results of 72h OD showed the statistical significance compared with the control group(P<0.01),and there are differences in half inhibited rates(IC50)which indicate that the different sensitivity between cells.The half inhibited rates(IC50)of PC9/R,H1975,H820,A549 IC50 are significantly larger than PC9,which has defined the PC9/R,H1975,H820,A549 are the resistance cells of gefitinib and PC9 are sensitive cell of gefitinib.Combining 0.25 mg/ml the aqueous extract of T.chinensis with different doses gefitinib(PC9/R,A549,H1975,H820 are 1,3,6,9,12,15,18,30 umol/L,PC9 are 0.01,0.02,0.04,0.1,0.5,1,5,10 umol/L)in different cells,the results present that the inhibition effect of combination are more significant than gefitinib(compared with gefitinib.P<0.05).The results indicate that the two medicines have synergistic effect,but when the dose of the gefitinib continue to increase the drug lethality does not rise significantly.2.Real-time PCR:Compared with gefitinib combination group significantly decreased the expression of EGFR(P<0.05)in A549.H1975.H820,PC9 and PC9/R.For PC9/R cells.the expression of STAT3 mRNA significantly decreased in combination group(P<0.05).For A549.PC9,PC9/R.cells.the expression of ERK1/2 mRNA significantly decreased in combination group(P<0.05).STAT3 mRNA in H1975.H820.PC9.A549 cells.ERK 1/2 mRNA in H820.H1975 cells kept equallv.Compared with blank control group,the expression of EGFR.ERK1/2,STAT3 mRNA all decreased significantly in AETC group(P<0.05)in 5 kind of cells.3.Western-blot:Combination group significantly decreased the expression of p-STAT3 and p-EGFR proteins in A549.H1975.H820,PC9.PC9/R(P<0.05).while STAT3 protein kept equally.In H1975,H820,PC9/R protein EGFR slightly decreased in combination group than other groups.while the expression kept equally in A549.PC9.Protein p-ERK1/2 expression decreased in H1975.H820.PC’9/R.A549(P<0.05).while kept equally in PC9.Conclusion 1.AETC in combination with gefitinib can increase the inhibition of human Lung cell lines effect than single drug.2.AETC probably overcomes gefitinib drug resistance and promotes the anti cancer effect by inhibiting the activation of EGFR and block its download signaling pathway as ERK1/2 and STAT3.