Study Of Capilliposide From Lysimachia Capillipes Restores Gefitinib Sensitivity In Human Non-small Cell Lung Cancer Cells With Acquired Gefitinib Resistance Mediated By T790M Mutation

Most non-small cell lung cancer(NSCLC)patients with gefitnib treatment would eventually develop epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)resistance,half of resistant patients carrying acquired T790M mutation.Previous studies showed that Lysimachia capillipes(LC)capilliposide have potential anti-cancer effects both in vitro and in vivo.In this study we investigated whether the combination treatment of LC capilliposide and gefitinib could restore the gefitinib sensitivity and the involved signaling pathways in NSCLC cells.IC50 values of five NSCLC cells lines with different sensitivity to gefitinib treated with LC capilliposide vary around the range of μg/mL.In combination with gefitnib,the geftinib-resistant PC-9-GR cells with acquired T790M mutation showed the significant increase of cell growth inhibition,with IC50 of gefitinib from 6.80±1.00 to 0.77±0.12μM.The median effect analysis showed the LC combined with capilliposide has good synergic effect with combination index(CI)value below 1.0.Combination treatment increaed the cell apoptosis induced by gefitinib,but did not change the cell cycle arrest in G0/G1 phase.Furthermore,we abserved that LC capilliposide significantly decreased the phosphorylation level of AKT signaling,compared with LC and gefitinib treatment alone.With siRNA slience of AKT in PC-9-GR,combination with LC capilliposide did not change the sensitivity of cells to gefitinib.In a xenograft model of PC-9-GR,LC capilliposide(15mg/kg/d)combined with gefitinib(50mg/kg/d)markedly enhanced tumor growth inhibition with a TGI of 109.3%.In groups of LC capilliposide or gefitinib treated alone,the TGIs are 22.6%and 56.6%,respectively,LC capilliposide can overcome the NSCLC cells’ resistance to EGFR-TKI by suppression of phosphorylation level of AKT signaling,suggesting that a LC capilliposide in combination with gefitinib may be a promising clincial therapeutic strategy to restore EGFR-TKI sensitivity in NSCLCs with an acquired T790M mutation.Part 1:Establishment and Characterization of EGFR-TKI gefitnib-resistant PC-9-GR cellsObjective:To establish and characterize the EGFR-TKI gefitinib-resistant PC-9-GR cells.Methods:PC-9 cells with 19Del muation of EGFR were under chronic suppression of gefitinib with increasing concentration.We first started to treat the PC-9 cells with 8.0nM of gefitnib,and then increased the dose step by step.When the cells can maintained in the dose of 8.0μM,we selected the subclones,and EGFR T790M muation detection was conducted.During the one year gefitinib-free maintenance,PC-9-GR was analysis by MTS assay,T790M mutation detection,cell growth curve and cell cycle analysis.Results:We found that the IC50 for gefitinib increased from 12.5nM in parental PC-9 cells to 8.6μM in PC-9-GR cells,which was appropriate 680-fold higher by using MTS assay.No obvious morphologic change of PC-9-GR cells was observed compared with parental PC-9 cells.EGFR T790M mutation was detected in PC-9-GR cells.Furthermore,we found that maintenance in gefitnib-free medium for one year did not change the gefitinib-resistance of PC-9-GR cells.However,cell cycle distribution slightly changed in PC-9-GR cells(PC-9-GR vs.PC-9:G0/G1 54.85±1.57%vs.49.03±0.84%;S 35.70±1.66%vs.36.75±3.02%;G2/M 9.45±0.56%vs.14.22±2.21%);.We also observed that the PC-9-GR has longer cell doubling time(41.0±3.1 vs 31.9±3.3 hours)with cell growth analysis,and the phosphorylations of EGFR,ART and ERK escalated obviously when compared to that of PC-9 cells by western blot analysis.Conclusion:EGFR-TKI gefitinib-resistant PC-9-GR cells was developed,the resistant cell line with acquired T790M mutation was stable and can be used in the gefitinib resistance studiesPart 2:Mechanisms of LC capilliposide restores gefitinib sensitivity in PC-9-GR cells with acquired gefitinib resistanceObjective:To investigate the mechanisms of LC capilliposide restores gefitinib sensitivity in PC-9-GR cells with acquired gefitinib resistance.Methods:Inhibitory effects of gefitinib and/or LC capilliposide were determined by MTS,median effect analysis was performed to test the synergic effect.Cell apoptosis and cell cycle were analyzed by flow cytometry.Western blot analysis and phospho-receptor tyrosine kinase array assay were performed to detect the relative levels of phosphorylation for kinase targets.siRNA transfection was used to evaluate the role of the key targets on the restoration of LC-induced sensitivity in resistant cells.Results:The potential effects of LC capilliposide on the growth of the five non-small cell lung cancer cell lines,including H460,H1299,H1975,PC-9 and PC-9-GR,in response to gefitinib treatment were examined.In combination with gefitnib,the geftinib-resistant PC-9-GR cells with acquired T790M mutation showed the significant increase of cell growth inhibition,with IC50 of gefitinib from 6.80±1.00 to 0.77±0.12μM.The median effect analysis showed the LC combined with capilliposide has good synergic effect with combination index(CI)value below 1.0.Gefitinib combined with low dose of LC capilliposide significantly increased the cell apoptosis induced by gefitinib in PC-9-GR cells with the total apoptosis of 34.6±2.0%(P<0.05),but did not change the gefitinib induced G0/G1 arrest,compared with groups of LC capilliposide or gefitinib treated alone.Moreover,we abserved that LC capilliposide significant decreased the phosphorylation level of AKT signaling,compared with LC and gefitinib treatment alone.With siRNA slience of AKT in PC-9-GR,combination with LC capilliposide did not affect the sensitivity of cells to gefitinib,no obvious change was observed in the group treated with gefitinib alone(P>0.05).Conclusion:LC capilliposide combined with geftinib could significanly inhibit AKT signaling and restores gefitinib sensitivity in PC-9-GR cells with acquired T790M resistant mutation in vitro.Part 3:Study on inhibitory effect of LC capilliposide on H460 xenograft tumors in vivoObjective:To investigate the therapeutic effect of LC capilliposide on gefitinib resistant PC-9-GR xenograft tumors in vivo.Methods:PC-9-GR cells were inoculated subcutaneously into the right thigh of 4-to 6-week-old BALB/C,Nu/Nu mice with weight of 18±2g.The mice were randomized into four groups when the average tumor volume reached 200mm3.The mice of each group were treated as following:(1)methylcellulose/Tween80 as vehicle control for 10 days;(2)gefitinib(50mg/kg/d)alone for 10 days;(3)LC(15mg/kg/d)alone for 10 days;and(4)gefitinib(50mg/kg/d)+LC(15mg/kg/d)for 10 days.The tumor volume and weight were measured twice a week.p-EGFR、p-AKT、Ki67、cleaved Caspase-3 proteins in xenograft tumor were analyzed by immunohistochemistry(IHC)assay.Results:Treatment with either LC capilliposide(15mg/kg for 10 days)or gefitinib(50mg/kg for 10 days)alone could inhibit in vivo PC-9-GR tumor growth with tumor growth inhibitions(TGIs)of 22.6%and 56.6%,respectively.LC capilliposide combined with gefitnib significantly increased the tumor growth inhibition with a TGI of 109.3%when compared to the treatment with gefitinib or LC capilliposide alone(P<0.05).The results of IHC assay showed that the combination treatment further decreased AKT phosphorylation and Ki67 expression;however,it did not cause an obvious change in EGFR phosphorylation when compared to each single treatment in the PC-9-GR xenograft tumors.In addition,we observed an increase of positive CC3 staining in the tumor specimens of the xenograft with combination treatment(P<0.05).Conclusion:LC capilliposide could inhibit the growth of gefitinib-resistant PC-9-GR xenograft and no obvious side effects were observed during the treatment.