| Objective:Cancers of the lung have long been the leading cause of cancer-related death all over the world.The largest subgroup of lung cancer is non-small cell lung cancer(NSCLC),occurring at the frequency of about 80%.Generally speaking,the therapeutic effect of NSCLC is far from satisfactory.Although great progress has been made in the chemotherapy,almost no more than 10 months median overall survival(OS)can be achieved even by the most effective platinum-based chemotherapeutic regimens.With deeper digging into the molecular events underlying the oncogenesis and progression of NSCLC,a kind of tyrosine kinase receptor,epidermal growth factor receptor(EGFR),became one of the landmark targets of NSCLC therapy.It forms dimers with other EGFR or other HER family members to get itself auto-phosphorylated at the key tyrosine residues.Subsequently,the phosphorylated EGFR further activates several downstream signaling pathways such as PI3K/AKT/mTOR,RAS/RAF/MAPK,JAK/STAT,which play the critical roles in regulating multiple cellular processes,including proliferation,survival and apoptosis.The constitutive activation of EGFR signaling path-way,caused by gene mutations or by gene amplification or both,has been demonstrated to have close connection with the initiation,progression and poor prognosis of NSCLC.EGFR activating mutations,majorly located in the tyrosine kinase domains and in the form of a base-pair deletion at exon 19 or a point mutation at exon 21,occur at about 20% of NSCLC patients.They enable the EGFR to activate,and thus induce the activation of downstream molecules.The first-generation EGFR tyrosine kinase inhibitors(EGFR-TKIs),gefitinib and erlotinib,designed to reversibly compete for the ATP binding sites and thus block EGFR-induced downstream signaling activation,have and are being extensively investigated in NSCLC treatment.A large number of studies showed gefitinib and erlotinib could significantly prolong median progression free survival(PFS)compared with carboplatin/paclitaxel in the subgroup of patients with EGFR mutation-positive tumors,together with much improved qualification of life and delayed deterioration of symptoms.However,more arduous efforts should be made since 30% of the EGFR mutation-positive subgroup cases are not responses to EGFR-TKIs.Therefore,it is important to explore the molecular mechanism of the sensitivity of tumor cells to EGFR-TKI,and it is of great significance for the rational selection of NSCLC drugs and the efficacy of drug treatment.Filamin A(FLNa),also known as actin-binding protein 280(ABP 280),has been associated with actin as cytoskeleton regulator.It was first identified as a non-muscle actin filament cross-linking protein.Subsequent reports revealed the importance of this protein in cell contraction and spreading.Filamins largely act as scaffolding molecules,facilitating protein–protein interactions and influencing protein cellular localization.Studies identified FLNa-binding proteins involved in cell signaling,overexpressed in multiple types of cancer.Recently its role in the tumor cell has become a hot research topic for FLNa’s involvement in cancer development.Professor Zhu has revealed knockdown the expression of FLNa can promote the proliferation,migration and invasion abilities of PC-9 cells.FLNa could increase the inhibitory effects of gefitinib on the proliferation,migration and invasion potential in PC-9 cells via increasing the inhibition of gefitinib on the EGFR activation and its downstream MAPK/ERK signal pathways.Erlotinib and gefitinib have the same quinazoline mother circus ring,the latent chain of gefinitib is not symmetry,while erlotinib is mirror symmetrical,the tiny difference on structure of them lead to the differences on blood plasma,distribution,metabolism,in vitro activity and toxicity.If the difference of the two drugs would cause different interaction with FLNa,thereby affecting the sensitivity to erlotinib of EGFR mutation positive non-small cell lung cancer,which needs further verification.The following experiments were performed in NSCLC cell line PC-9 cells,which were with exon 19 deletion mutation:(1)Stably transfected cell line with knockdown of FLNa and control cell line were established and verified by Western blot.(2)Effects of erlotinib on the biological behavior of stable PC-9 cell lines,including proliferation,migration and invasion,were examined by MTS,wound healing assay and transwell cell invasion assay.(3)Western blot was used to detect the level of phosphorylation of EGFR,ERK signal molecule after the effects of erlotinib.Methods:1 Establishment of stably transfected cell lines,stably transfected cell line with knockdown of FLNa and control cell line were established and verified by Western blot.2 MTS assay was used to measure the OD values and to calculate the growth inhibition rate and the half inhibitory concentration(IC50)of erlotinib.3 Wound healing assay was used to examine the migration abilities of stably transfected cell lines affected by erlotinib.4 The invasion assays was used to examine the invasion ability of stably transfected cell lines affected by erlotinib.5 Western blotting was used to examine the levels of Filamin A,p-EGFR,EGFR,p-ERK,and ERK.?-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 PC-9/FLNa(KD)cells(0.25±0.01)were significantly lower than in PC-9/ctrl group cells(0.87±0.01)(P<0.01).2 The sensitivity to erlotinib in stably transfected cell lines2.1 The IC50 value of erlotinib in stably transfected cell linesThe cell growth inhibition rates of PC-9/ctrl cells at different concentrations(0,0.01,0.02,0.04,0.08,0.16,0.32,0.64μmol/L)of erlotinib were markedly higher than those of PC-9/FLNa(KD)cells(P<0.05,respectively).The IC50 value of PC-9/FLNa(KD)cells(0.39±0.01)were significantly higher than that of PC-9/ctrl cells(0.08±0.01)(P<0.01).2.2 Migration abilities of stably transfected cell lines treated with or without erlotinibMigration rates of PC-9/FLNa(KD)cells without erlotinib for 6h,12 h and 18h(16.49±0.86%,43.32±0.95%,50.00±0.00%)were significantly higher than that of PC-9/ctrl(11.58±1.50%,28.68±1.48%,50.00±0.00%)(P<0.05,respectively).Migration rates of PC-9/FLNa(KD)cells with erlotinib(0.04μmol/L)exposure for 6h,12 h and 18h(12.92±1.44%,30.35±1.07%,49.98±0.02%)were significantly higher than that in PC-9/ctrl cells(6.36±0.99%,18.48±1.10%,24.22±0.05%)(P<0.05,respectively).2.3 Invasion abilities of stably transfected cell lines treated with or without erlotinibThe average invasion cell numbers of PC-9/FLNa(KD)cells without erlotinib(125.00±5.72)were significantly more than that in PC-9/ctrl cells(63.00±5.46)(P<0.01).The average invasion cell numbers of PC-9/FLNa(KD)group with erlotinib(0.04μmol/L)exposure(86.00±4.09)were significantly more than that in PC-9/ctrl group(29.00±3.21)(P<0.01).3 Levels of EGFR phosphorylation and the downstream signal molecules in stably transfected cell lines treated with erlotinibThe levels of different proteins in stably transfected cells were detected by Western blot after the treatment of erlotinib(0.01μmol/L)for 0,2 and 4h.After the treatment of erlotinib(0.01μmol/L)for 0,2 and 4h,the levels of p-EGFR in PC-9/FLNa(KD)group(0.37±0.01;0.31±0.01;0.28±0.00)were significantly higher as compared with PC-9/ctrl group(0.33±0.01;0.03±0.00;0.11±0.00)(P<0.05,respectively).The levels of p-ERK in PC-9/FLNa(KD)group(0.98±0.00;0.57±0.00;0.79±0.01)were significantly higher as compared with PC-9/ctrl group(0.89±0.01;0.25±0.01;0.45±0.01)(P<0.05,respectively).Conclusion:1 Down-Regulation of FilaminA expression can promote the proliferation,migration and invasion abilities in PC-9 cells,Filamin A may affect biological behaviour of PC-9 cells through regulating EGFR phosphorylation and its downstream MAPK/ERK signal pathways.2 FilaminA improved sensitivity to erlotinib in PC-9 cells via increasing the inhibition on the EGFR activation and its downstream MAPK/ERK signal pathways. |
