| Objective: Lung cancer is the leading cause of cancer-related death in China.It ranks first in the morbidity and mortality of male malignancies,while,the morbidity in female tumor was second and the mortality still stay in a high position without going down.Non-small cell lung cancer(NSCLC)was the most common type in lung cancer,which accounts for 85%.The main treatments include surgery,chemotherapy,radiotherapy,molecular targeted therapy and immunotherapy.In recent years,although the level of chemotherapy and surgical treatment have been improved,the 5-year survival rate is only increased to 15%.The molecular targeted therapy which targeting inhibits the tumor proto-oncogene or its signing pathway could exert inhibit tumor cells and reduce the damage on normal cells has created a new field in lung cancer treatment and brings the gospel for cancer patients.Epidermal growth factor receptor(EGFR)is a kind of tyrosine kinase active receptor(RTK),which belongs to ErbB family.Upon ligand binding,such as epidermal growth factor(EGF),EGFR family proteins dimerize by receptor homo-dimerization and subsequently auto-phosphorylated at the key tyrosine residues.Activated EGFR family receptors trigger a series of downstream signaling pathways,such as MAPK/ERK,PI3K/AKT pathways,JAK/STAT pathways,which regulating cancer proliferation,invasion,metastasis and angiogenesis.Studies have reported that EGFR activating mutations was associated with poor progression and poor prognosis of NSCLC patients.The most common EGFR mutations are deletions in exon 19 and point mutations in exon 21(L858R substitution mutations),occur at10 ? 15% of NSCLC patients in Western population and 40% of NSCLC patients in Asian population respectively.The molecular targeted drugs weresuccessfully applied in clinical practice.The first-generation epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs),sush as gefitinib and erlotinib have been widely used in first-line treatment of advanced NSCLC patients with EGFR activating mutations,which could significantly prolong median progression free survival(PFS)and improve qualification of life compared with platinum-based double chemotherapy.However,most patients ultimately developed disease progression after about 9~14 months.The most common acquired resistance mechanisms is either T790 M mutation that increases the affinity for ATP and reduces the efficacy of inhibitors.Otherwise,it induces MET gene amplification,small cell lung cancer transformation,EGFR mutation gene loss.In addition,about 30% of patients showed primary resistance to EGFR-TKIs.Considering the drug resistance,the second generation of imatinib and the third generation of Osimertinib(AZD9291)was researched and developed.As a novel oral drug,potent and selective irreversible inhibit the EGFRm+ sensitizing and T790 M resistance mutants,the effect is poor for the wild-type EGFR.Thus,we need explore the problem of the sensitivity of NSCLC patients to EGFR-TKIs,in order to offer some prompts for sequential treatment.Filamin A(FLNa)is a non-muscle actin-binding protein,which located in the cytoplasm.FLNa including an actin binding domain at the amino end and a rod-like domain consisting of 24 tandem repeats,the multiple layer structure of the β-sheet in the domain of rod-like molecules provides an interface for protein and protein interaction.FLNA could change oncogenic microenvironment in the manner of regulate signal transduction molecules.Currently,Zhu et al have found that knockdown the level of FLNa expression could accelerate the proliferation,migration and invasion of PC-9cells and decrease of the sensitivity of PC-9 cells to the first generation of EGFR-TKIs.However,after a period of treatment,patients often appear problems of drug resistance,in the light of the most common mechanism--T790 M mutation,the third-generation inhibitors AZD9291 was developed,its mono-anilino pyrimidine compound is structurally andpharmacologically distincts from other EGFR-TKIs.EGFR-TKIs with different structures have dissimilar mechanisms of mutual effect with FLNa,its affect to the cell sensitivity is unknown.Thus,we will learn and investigate the two points mentioned above.The following experiments were performed in NSCLC cell line H1975 cells,which were with EGFR twenty-first exon sensitive mutation and T790 M resistance mutation.Construction of stably transfected cell lines with knockdown of FLNa.Examining the biological behavior of transfected cell lines,including proliferation,migration and invasion,were examined by MTS,Colony formation assay,wound healing assay and Transwell cell invasion assay.Western blot was used to detect EGFR phosphorylation levels and variety of its downstream signal molecules after the effects of AZD9291.To explore FLNa influences to sensitivity of third generation EGFR-TKIs in lung adenocarcinoma H1975 cell with EGFR T790 M mutation.Methods:1 Proving the stably transfected cell line with knockdown efficiency of FLNa by Western blotThe lung adenocarcinoma H1975,H1975/FLNa(KD)and H1975/ctrl cells which our laboratory reserves were seeded into 35 mm dishes,then culture the cell with 1640 medium until plates were fully attached by the cells.Total proteins were extracted from cells.Using the western blot to examine the levels of FLNa,and ?-actin was used as a loading control.2 MTS assay was used to examine the proliferation abilities in stably transfected cell linesH1975/FLNa(KD)and H1975/ctrl cells were seeded in 96-well plates,respectively.After culturing for 24 hours,cells were treated with different concentrations of AZD9291(0,2,4,8,16μmol/L)and continue to grown for48 h,then add MTS to measure the OD values and calculate the growth inhibition rate and the half inhibitory concentration(IC50)of AZD9291.3 Plate cloning method was used to examine the formation of cell wallH1975/FLNa(KD)and H1975/ctrl cells were seeded in 35 mm dishes,with or without AZD9291(1μmol/L)for 2 weeks,then using microscope after hematoxylin staining to observe and count the number of colony formation.4 Wound healing assay was used to examine the migration abilities of stably transfected cell linesH1975/FLNa(KD)and H1975/ctrl cells were seeded in 24-well plates,respectively.After growing for 24 h,cells were scratched a sterile pipette tip,then treated with or without AZD9291(1μmol/L),cells were hardly changed.So,treated with or without AZD9291(2μmol/L),Images of cells at the same field were taken until the closure of the scratch.We measured width variation and calculated cell migration rate.5 The invasion assays was used to examine the invasion ability of stably transfected cell linesThe invasion assays were carried out using Transwell chamber with 10 mm diameter and 8 ? m pore size polycarbonate membrane coated with matrigel.H1975/FLNa(KD)and H1975/ctrl cells were seeded into upper chamber with serum-free 1640 medium with or without AZD9291,respectively.Lower chamber was added into 10% serum 1640 medium.After incubated for 24 hours,the invaded cells were fixed and stained.6 Western blotting was used to examine change of cell signaling pathway molecular activityH1975/FLNa(KD)and H1975/ctrl cells were seeded into 35 mm dishes with 1640 medium culture until to full,After the treatment of AZD9291(1μmol/L)in different time(0,2,4,8hour),total proteins were extracted from cells.Western blot was used to examine the levels of p-EGFR,EGFR,p-ERK,ERK and FLNa,and ?-actin was used as a loading control.Results:1 Detecting the expression of Flamin A in stably transfected cell linesThe relative levels of FLNa protein in H1975/FLNa(KD)cells(0.34±0.01)were significantly lower than in H1975/ctrl group cells(0.91±0.02)(P<0.01).2 The IC50 value of AZD9291 in stably transfected cell linesThe cell growth inhibition rates of H1975/FLNa(KD)cells at different concentrations of AZD9291 were markedly higher than those of H1975/ctrl cells(P<0.05,respectively).The IC50 value of H1975/FLNa(KD)cells(2.17±0.19)were significantly lower than that of H1975/ctrl cells(6.09±0.11)(P<0.01).3 Colony forming abilities of stably transfected cell linesColony formation rates of H1975/FLNa(KD)cells without AZD9291 for 2 weeks(91.18±3.27%)were significantly lower than that of H1975/ctrl(143.26±6.49%)(P<0.01,respectively).Colony formation rates of H1975/FLNa(KD)cells with AZD9291(1μmol/L)for 2 weeks(7.95±2.02%)were significantly lower than that of H1975/ctrl(21.07±3.21%)(P<0.01,respectively).4 Migration abilities of stably transfected cell lines treated with or without AZD9291Migration rates of H1975/FLNa(KD)cells without AZD9291 for 6h,12 h and 18h(5.21±0.85%,12.30±0.32%,19.09±0.66%)were significantly lower than that of H1975/ctrl(13.79±1.88%,33.49±1.92%,50.00±0.00%)(P<0.01,respectively).Migration rates of H1975/FLNa(KD)cells with AZD9291(2μmol/L)exposure for 6h,12 h and 18h(4.07±0.66%,9.26±1.48%,13.07±1.35%)were significantly lower than that in H1975/ctrl cells(7.93±1.05%,21.90±1.50%,33.90±1.27%)(P<0.01,respectively).5 Invasion abilities of stably transfected cell lines treated with or without AZD9291The average invasion cell numbers of H1975/FLNa(KD)cells without AZD9291(127.80±4.49)were lower than that in H1975/ctrl cells(221.60±5.86)(P<0.01).The average invasion cell numbers of H1975/FLNa(KD)group with AZD9291(1μmol/L)exposure(29.00±2.24)were lower than that in H1975/ctrl group(70.20±2.39)(P<0.01).6 Levels of EGFR phosphorylation and the downstream signal molecular activity in stably transfected cell linesThe levels of different proteins in stably transfected cells were detectedby Western blot after the treatment of AZD9291(1μmol/L)for 0,2,4 and 8h.the levels of p-EGFR in H1975/FLNa(KD)group(0.30±0.02;0.08±0.02;0.10±0.02;0.01±0.00),which were significantly lower as compared with H1975/ctrl group(0.36±0.02;0.44±0.01;0.16±0.01;0.06±0.01)(P<0.05,respectively).The levels of p-ERK in H1975/FLNa(KD)group(0.90±0.04;0.31±0.00;0.28±0.01;0.18±0.06)were significantly lower compared with H1975/ctrl group(1.11±0.09;1.42±0.02;1.12±0.12;1.01±0.11)(P<0.05,respectively).Conclusion:1 Down-Regulation the expression of Filamin A can increasing the inhibitory effects of AZD9291 on the proliferation,migration and invasion abilities in lung adenocarcinoma H1975 cell with EGFR T790 M mutation.2 Down-Regulation the expression of Filamin A can decreasing EGFR phosphorylation and its downstream MAPK/ERK signal pathways,improve sensitivity of third generation EGFR-TKI AZD9291 in H1975 cell. |
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