?-catenin Enhanced The Sensitivity Of NSCLC Acquired TRAIL Resistant Cell Line H460-TR

Part ? Construction and identification of acquired TRAIL-resistant cell line(H460-TR)Objective To investigate the acquired-TRAIL resistance lung cancer cell line H460-TR,we compared the susceptibility of the acquired TRAIL resistance lung cancer cell lines and parental cell line H460,and explored the molecular mechanism of TRAIL acquired resistance.Methods To construct the TRAIL acquired resistance lung cancer cell line H460-TR,the parental cell H460 was induced from low concentration to high concentration.Exploring the differences of TRAIL sensitivity between the sensitive and acquired resistance cell lines,CCK-8 and flow cytometry were performed.The expression of related proteins in extrinsic apoptosis and intrinsic apoptotic signaling pathway were detected by western blot.The expression of death receptors in H460 and H460-TR cells was detected by quantitative qRT-PCR and western blot.The expression of death receptors on the surface of the cell membrane was detected by indirect immunofluorescence staining by flow cytometry.Extracting the cell membrane protein of H460 and H460-TR,then detected the expression levels of death receptors by western blot.Results The morphological of acquired resistance cell line H460-TR and parental cell line H460 had no significant changes by optical microscopy.However,after inductio n by TRAIL,the cell death was more pronounced.Detection of TRAIL toxicity of acquired resistance and sensitive cell lines by CCK-8 method,it showed that the survival rate of H460 cells declined with the increase of dose,while H460-TR showed relative tolerance.Similarly,in the flow cytometry test,the apoptotic rate of H460 was significantly higher than that of H460-TR induced by the same dose of TRAIL and the same time.As a result,the acquired TRAIL resistance cell line H460-TR successfully constructed.There was no significant difference in the expression of related protein in the death-induced signal complex(DISC),but the expression of apoptotic antagonist cFLIP was increased in the acquired resistance cell lines.The anti-apoptotic Mcl-1 expression in drug-resistant cell lines increased by the detection of intrinsic apoptotic pathways.The expression of death receptors on the surface of H460 cells was gradually increased when induced by TRAIL,while in the H460-TR cells,the surface membrane of the H460-TR cells was not changed significantly.Conclusion The acquired TRAIL resistance cell lines H460-TR was constructed successfully.The expression of apoptotic antagonist cFLIP and anti-apoptotic Mcl-1 which increased in extrinsic and intrinsic apoptotic signaling pathway were also important for the TRAIL acquired resistance in H460-TR.At the same time,the expression level of death receptors on the surface of the cell membrane between sensitive and acquired resistant cell lines is a key factor affecting TRAIL-acquired resistance.Part? The mRNA expression profiles in TRAIL-resistant cell lines and TRAIL-sensitive cell lineObjective Investigate the differences in gene expression profiles of TRAIL-sensitive cells,primary drug-resistant cells and acquired TRAIL-resistant cell lines in non-small cell lung cancer.Methods The TRAIL sensitive cell H460,the primary resistant cell A549 and the acquired TRAIL-resistant cell line H460-TR were selected to screen the differentially expressed genes of TRAIL drug sensitivity by using the gene expression profile chip.Results The mRNA of H460 cell line,A549 cell line and H460-TR cell line were extracted to cDNA respectively.After the original data obtained by chip scanning was normalized,the data set of the difference gene was obtained by Fold-change and T test.A total of 5047 differentially expressed genes between A549 cells and H460 cells were screened,while 1338 differentially expressed genes between H460-TR cells and H460 cells were found.The difference gene was analyzed by GO enrichment analysis,combined with the KEGG website to screene out the difference gene and biological pathway analysis by combining with the KEGG website and drew the KEGG path map.The results of apoptosis signal pathway included different expressied genes between A549 cells and H460 cells and different expressed genes between H460-TR cells and H460 cells.Conclusion Multi-target and multi-gene changes were related in the resistance process regardless of the primary drug resistance and induction of TRAIL-resistant.Further in-deep investigation of the signaling pathways and related genes function,the interpretation of the exact molecular mechanisms of TRAIL resistance will be benefit to provide new targets and ideas to targeted drugs.Moreover,a new perspective will be prepared for the specific molecular mechanisms of TRAIL resistance in clinical application.Part III Study on TRAIL sensitivity regulated by TRKA phosphorylated ?-cateninObjective Investigate the expression of ?-catenin in acquired TRAIL-resistant cell lines and to analyze the effect of ?-catenin expression on susceptibility of sensitive and acquired resistant cell lines.The effect of ?-catenin on TRAIL sensitivity of cells was investigated by detecting the membrane protein level of DR4/5.Investigate the mechanism of ?-catenin reversing TRAIL resistance.Methods We screened out the difference gene which interacted with ?-catenin.Then,we investigated the correlation between TRKA-regulated ?-catenin and the sensitivity of TRAILS in drug-resistant cells by TRKA-specific small molecule inhibitors.qRT-PCR and Western blot were used to detect the expression of ?-catenin in H460 and H460-TR cells.Constructed P-catenin gene silencing lentivirus system and transfected the over-expression plasmid into H460/H460-TR cells,then verified the silencing or overexpression efficiency by RT-PCR and Western blot.Changes of susceptibility to TRAIL with ?-catenin silencing or overexpression were dected with CCK-8 and flow cytometry.The expression of caspase family in the downstream signal transduction pathway was also detected.To confirme that P-catenin affected the expression of death receptor on the cell membrane and the different sensitivity of TRAIL,we detecedt the expression of death receptor and the expression of membrane protein after silencing or over-expression of ?-catenin.Results By analyzing the gene interacting with ?-catenin and performing qRT-PCR,we found that TRKA was highly expressed in H460-TR.The use of TRKA kinase small molecule inhibitors in combination with TRAIL in acquired TRAIL-resistant H460-TR cells can reverse the sensitivity of acquired TRAIL-resistant tolerance.The occurrence of ?-catenin phosphorylation could be inhibited by using TRKA-specific small molecule inhibitors and stabilized the cell membrane to avoid phosphorylation of ?-catenin from the cell membrane,dissociation into the cytoplasm and degradation,resulting in TRAIL sensitivity changes.The gene and protein expression of ?-catenin were significantly decreased in H460-TR,with that of H460.Silencing or overexpressing ?-catenin in Hela cells was confirmed effectively by the expression of gene and protein levels.The construction of ?-catenin gene silencing lentivirus system was also constructed by the interference sequences.Using CCK-8 and flow cytometry to detect cell apoptosis showed that TRAIL sensitivity decreased when?-catenin was silenced in H460.In contrast,the TRAIL sensitivity of the resistant cell lines H460-TR increased.with the overexpression of ?-catenin.The detection of apoptosis-related protein expression results showed that ?-catenin silencing or overexpression decreasing or increasing TRAIL sensitivity was affected by the activation of caspase family.While the ?-catenin gene silencing or overexpression,the death receptor gene and protein levels were positively correlated by qRT-PCR and western blot detection.In particular,the expression of the death receptor on the cell membrane was associated with the expression of ?-catenin after the induction of TRAIL.Conclusion By using TRKA kinase small molecule inhibitors combined with TRAIL in TRAIL-resistant H460-TR cells,it was possible to reverse the sensitivity of TRAIL-resistant cell lines.TRKA was able to dissociate into the cytoplasm by phosphorylation of ?-catenin and degrade it from the cell membrane.The occurrence of acquired TRAIL-resistant cells was associated with a decreased expression of?-catenin,whereas the overexpression of P-catenin increased the sensitivity of acquired TRAIL-resistant cell H460-TR to TRAIL.The effect of P-catenin on the sensitivity of TRAIL drugs was investigated by the silence or overexpression of P-catenin.It was found that ?-catenin was able to up-regulate the expression of death receptors on the surface of cell membrane.When the ligand and receptor combined more effectively,the activity of downstream of the apoptotic molecule caspase family increased.Thus,promoting the occurrence of apoptosis and reversing the recurrence of lung cancer cells TRAIL tolerance.