Establishment Of A Gefitinib Resistant Cell Line NCI-H1975/GR And Investigation On The Drug Resistance Mechanism

Objective：Currently surgical resection is the most effective way to therapynon-small cell lung cancer (NSCLC), which is the most common type in lung cancers.However, when found to be suffering from the cancer, a lot of patients are already in theadvanced stage of clinical stage, some of whom have lost the opportunity to beoperated, moreover the patients having accepted the surgical operation require adjuvantchemotherapy too. Therefore, chemotherapy is the widely used approach for treatingNSCLC clinically, yet as a result of low specificity and more side effects, theapplication of traditional chemotherapeutics is restricted in treatment of NSCLCclinically. Overcoming the shortcoming of traditional chemotherapy, the moleculartargeted therapy is extensively applied in the file of tumor therapy. Epidermal growthfactor receptor-tyrosine kinase inhibitor (EGFR-TKI), represented by thegefitinib(ZD1839, iressa), is the most successful molecular targeted medicine in treatingNSCLC. EGFR-TKI can compete against EGF for the highly conservative ATP bindingsite in the structural domain of EGFR, selectively controls the activity of EGFR proteintyrosine kinase, block EGFR and the startup of its transduction system, make thetransduction system of RAS/RAF/MAPK inactive and inhibit the proliferation of tumorcell. It is revealed that NSCLC which is sensitive to EGFR-TKI contains the mutationsin EGFR18,19,21exons, and these mutations are more frequently seen inadenocarcinomas from Asian, female and non smoker. On the other hand, NSCLC withEGFR-TKI drug resistance normally contains mutation in EGFR20, K-RAS or B-RAFexon. It is noticeable that with the extending of EGFR-TKI medication, some NSCLCswhich were sensitive to EGFR-TKI have been generated the acquired drug resistancebring therapy of the drug to failure. Therefore, exploring the mechanism of acquireddrug resistance of NSCLC to EGFR-TKI has been the urgent problem clinically. The acquired drug resistance mechanism of traditional chemotherapeutics contains theincreasing expression of drug resistance related protein, dysfunction of mismatch repair(MMR), the increasing excretion and deintoxication of cell drugs and the change ofdrug target. But it is difficult to explain the acquired drug resistance mechanism ofEGFR-TKI by using that of traditional chemotherapeutics. It has been reported that theacquired drug resistance mechanism of EGFR-TKI of NSCLC are mainly as follows,secondary mutation of EGFR20exon which invokes the change of EGFR conformationand makes the EGFR-TKI lose the target, K-RAS and (or) B-RAF gene exon mutationwhich enhances the autoactivation or sensitivity of molecular in the downstream of thesignal transduction pathway, epithelia to mesenchyma transition (EMT), lost ofepithelial features of cancer tissue, which makes the drug lose the target, and METgene amplification. The occurrence of aforementioned mechanism, such as genemutation, is usually related to the abnormal DNA repair function, such as the abnormalMMR. To study these mechanisms, the EGFR-TKI acquired drug resistance model invitro is necessary, but there are few these models in China. In this research, weestablished the EGFR-TKI acquired drug resistance cell line NCI-H1975/gefitinibresistance (NCI-H1975/GR) of lung adenocarcinoma in vitro, compared the changes ofEGFR, P-EGFR, K-RAS, B-RAF，E-cadherin and MMR protein expressions betweendrug resistance cell line and parent cell line, in order to provided data for investigatingEGFR-TKI acquired drug resistance mechanism of NSCLC.Method:1. Establishment of EGFR-TKI acquired drug resistance cell lineNCI-H1975/GRThe human lung adenocarcinoma cell line NCI-H1975was bought from theinstitute of basic science of Chinese academy of medical science. The cell lineNCI-H1975/GR is established by gradually increasing density and intermittent revulsionbased on gefitinib. The drug resistance index of cell line is detected by MTT. Thechange of cellular morphologies of parent and drug resistant cell lines before and aftermedicine is observed under inverted microscope. The growth curve and populationdoubling time of parent and drug cell lines are depicted with cytometry, and the cellulardistribution is analyzed with the cytometry too.2: Detection of EGFR-TKI acquired drug resistant cell line NCI-H1975/GRThe protein expressions of EGFR, P-EGFR, K-RAS, B-RAF， E-cadherin andMMR of drug resistant and parent cell line were detected immunocytochemically.Discuss the EGFR-TKI acquired drug resistance mechanism of NSCLC by comparing the protein expression difference between drug resistance and parent cell lines.Results:1. The NCI-H1975/GR, one EGFR-TKI drug resistant cell line of humanlung adenocarcinoma with drug resistance index being1.282.to was made up afterinduction of six monhs.It is found that there is cellular morphology difference of NCI-H1975/GR toNCI-H1975. The most of cells in NCI-H1975/GR appear as long spindle, while the restof cells are round or spindle. The population doubling time of NCI-H1975/GR is7.4hlonger than that of NCI-H1975(P<0.05). The results observed by flow cytometerreveal that81.11%cells of NCI-H1975/GR were G1and G0，14.63%were in phase Sand other4.26%in phase G2, but49.80%cells of NCI-H1975were G1and G0,42.20%were in phase S and other8.00%in phase G2.2: Immunocytochemical results(1) The cells in both NCI-H1975and NCI-H1975/GR had EGFR and P-EGFRprotein expressions in the cytoplasm and nucleus, while the expression in theNCI-H1975/GR was stronger than that in NCI-H1975.(2) The cells in both NCI-H1975and NCI-H1975/G had K-RAS expression in thecytoplasm, but the expression in the NCI-H1975/GR was weaker than that inNCI-H1975.(3) The cells in both NCI-H1975and NCI-H1975/GR had B-RAF expressions inthe cytoplasm, but the expression in the NCI-H1975/GR was weaker than that inNCI-H1975.(4) The cells in both NCI-H1975and NCI-H1975/GR had E-cadherin expressions,but the expression locations were different between them. Of NCI-H1975/GR, theE-cadherin expression decreased in membrane, while increased in cytoplasm, comparedto that of NCI-H1975.(5) The cells in both NCI-H1975and NCI-H1975/GR had mismatch repair proteinhMSH2expressions in nucleus, while the expression in NCI-H1975/GR was weakerthan that in NCI-H1975.Conclusion:1．The protein expressions of both EGFR and P-EGFR in gefitinibresistant cell line increase.2．The K-RAS and B-RAF protein expressions decrease in EGFR-TKI resistantcell line, which might be related to slow growth of the cells in EGFR-TKI resistant cellline.3．The E-cadherin expression, as a marker for epithelia decreases in EGFR-TKI resistant cell line, and this finding indicates that one of reasons by which EGFR-TKIresistance development is loss of the epithelial differentiation.4. MMR，hMSH2protein expressions in gefitinib resistant cell line decrease, theabnormal mismatch repair gene function may be one of the reasons for development ofEGFR-TKI resistance in NSCLC.