| BackgroundLung cancer is the most common clinical disease cause death in recent years, showeda higher morbidity and mortality. More than50%of lung cancer patients were diagnosedbefore progression and metastasis. Regardless of the treatment, the5-year average survivalrate is only13-20%, so further study of the incidence of lung cancer as well as themechanism of resistance is still very necessary.Gefitinib as an epidermal growth factor receptor tyrosine kinase inhibitors, can becompetitive to the cell surface of epidermal growth factor receptor tyrosine kinase(EGFR-TK) ATP binding points on the catalytic domain, thereby inhibiting EGFRphosphorylation and activation of downstream signaling pathways, has been approved bythe FDA to continue to deteriorate with therapy after chemotherapy for advancednon-small cell lung cancer (NSCLC). Gefitinib as a targeted drug to improve the efficacyand quality of life of advanced non-small cell lung cancer showed some advantages,especially for characteristic mutations of EGFR kinase domain (exon19deletion and exon21L858R mutation) of the patients. These patients with the duration of treatment,eventually the inevitable emergence of drug resistance, lead to the progress of the disease.Clarify the mechanisms of gefitinib resistant during treatment, to explore synergies withinhibition of resistance method for better play gefitinib gefitinib pharmacodynamicspotential to further improve lung treatment of the clinical level, is of great significance.Studies over the last few years have identified two different EGFR-TKI resistancemechanisms, a secondary mutation in EGFR, EGFR T790M, and amplification of the METoncogene, which have been reported in～50%and20%, respectively, of patients acquiringresistance to EGFR-TKIs. However, present study shows that some patients with NSCLCEGFR gene did not existed secondary mutations, is still secondary resistance togefitinib.There are a small number of patientsT790M mutation can be detected beforetreatment with gefitinib, suggesting that gefitinib resistance can not be explained by a sigletheory.Study found the mechanism that tumor cell resistance to drug-induced apoptosis iscomplex, of which a large part is due to the tumor cells promote survival genemutation.However, it usually takes a long time to get these drug-resistant genotypes. So these cells may need some non-gene mutation mechanisms before getting resistantgenotypes. Therefore, we proposed that, temporary or persistent resistance mechanisms inearly time of drug treatment may have begun to play a role.Of the current study it is generally believed that the tumor is not only limited toabnormal gene expression in malignant cells, but also include interaction with the tumorcells by the host into fibroblasts, endothelial cells and immune cells, like tumormicroenvironment. The interaction between each other will determine the fate of tumor andalso the response of tumor cells to drug. Cytokines in the tumor microenvironment caninduce gene transcription or activation of post-transcriptional modification, thus reducingthe sensitivity to the drug. This kind of mechanisms of resistance is transient and reversibletherefore could be a target for the eradication of minimal residual disease. Blockingmicroenvironment mediated resistance may prevent recurrence or to extend the time ofrecurrence.Fibroblasts commonly found in lung cancer cell microenvironment, able to expressand secrete large amounts of cytokines and matrix components so as to promote theproliferation of tumor cells to survive. Therefore, the purpose of this study is to researchthe influence of fibroblasts on the sensitivity to gefitinib of NSCLC cell lines HCC827andexplore the mechanisms. Looking forward to block the microenvironment mediatedresistance in the early treatment therefore to achieve the greatest extent prevent recurrenceor to extend the time of recurrence and improve survival of patientsperiod.Part1The influence of coculture with fibroblasts on the sensitivity to gefitinib ofNSCLC cellsSuccessfully isolated from the tumor tissue of the primary fibroblast cells, and wereidentified by Vimentin markers. Through Transwell technical to study the influence ofprimary fibroblasts and fibroblast cell line MRC-5on the sensitive to gefitinib of NSCLCcell lines HCC827. By MTT assay we found that under co-culture conditions two kinds offibroblasts both can weaken the the tumor cell growth inhibition rate induced by gefitinib(P <0.05). Within48h prior co-culture, no significant effect observed on the degree ofgefitinib sensitivity.Part2The mechanism of reduced sensitivity to gefitinib of HCC827co-cultured withfibroblastThe main mechanism of Gefitinib is through inhibition of EGFR phosphorylation thus blocking the transfer of downstream cell growth signal. In this study, the apoptosis and cellcycle under cultured alone and co-cultured were determined by flow cytometry. The resultof flow cytometry showed that the rate of tumor cell apoptosis in culture conditionsreduced20%compared to cultured alone.(P <0.05). And also found that gefitinib couldreduce the rate of G0/G1phase of tumor cells cultured alone, and the co-culture conditionscan reduce this kind of arrest. By Western blotting also found that under co-culturedcondition tumor cells expression higher level of p-Akt and p-ERK while the EGFRphosphorylation status remain unchanged. Therefore, we infer fibroblasts induced tumorcells gefitinib response reduced is likely to be due to the downstream signaling proteinsbypass activation, thus promoting tumor cell survival, to reduce the drug-induced apoptosis.Also found that MET amplification was not the primary mechanism of reduced sensitivityto gefitinib under the coculture condition. Further, in the present experiment also studiedthe role of fibroblast MRC-5culture medium in the reduction of gefitinib sensitivity. Wefound the soluble factors secreted by fibroblasts caused the reduced sensitivity of tumorcells to drug. So we conclude that fibroblasts secrete soluble factors involved indownstream pathway bypass activation while the EGFR signaling pathway is blocked.Part3Screening of differentially expressed genes and bioinformatic analysis ofCo-culture HCC827under different treatment conditionsIn order to study the mechanisms involved in the sensibility of tumor cells to gefitinibof fibroblasts secrete soluble factors, Four groups Affymetrix Primeview whole genomemicroarray screening of tumor cells the HCC827under co-cultured and gefitinib treatmentwas performed. By high-throughput analysis we found that the number of gene expressionchanged under culture alone and co-cultured conditions were642(4vs2group).According the result of GO analysis and Pathway analysis, these genes are involved in awide range of functions, including cell cycle regulation, cell growth, apoptosis, signaltransduction; also a wide range of signaling pathways such as MAPK, P53, cell cycle,focal adhesion, Wnt pathway and s TGF-beta pathway. These results contribute to theglobal understanding the response to gefitinib of tumor cells under different cultureconditions. By literature search and gene function, we screened e eight gene for furtherstudy. However, whether they are key genes involved in drug response and their role in themechanism of different response to gefitinib under co-culture condition still needs furtherexperimental study. Part4Verification of the different genes and influence of over-expressed AURKA onthe gefitinib sensitivityAURKA plays an important role in normal mitosis. In order to study whether itparticipate in different response mechanism, we constructed AURKA over-expressionplasmid. Then gefitinib sensitivity was measured to analyze whether it cause the reducedsensitivity. The results showed that AURKA over-expression can reduce the gefitinibsensitivity, but still need further RNA interference to verify. |
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