| Background and Objective:Non-small cell lung cancer (NSCLC) is the most prevalent malignancy in bothmorbidity and mortality. The main therapeutic method of NSCLC is surgical resectioncombined with adjuvant chemotherapy. However, when found to be suffering from theNSCLC, more than half of the patients have already been in the advanced stage, andsome have lost the precious opportunity of surgery. Even if having accepted the surgicaloperation, the patients often need adjuvant chemotherapy too. Therefore, thechemotherapy is the most widely used approach for clinical treatment of NSCLC.However, the traditional chemotherapy has many limitations in clinicalapplications due to poor specificity and severe side effects. In order to overcome theshortcomings of traditional chemotherapy, molecular targeting individualized treatment,in which the appropriate drug was chosen for the key tumor-specific genetic targets, hasbe becoming the main trend in the clinical oncology. Epidermal growth factor receptortyrosine-kinase inhibitors (EGFR-TKI), such as gefitinib and erlotinib, are the mostwidely used molecular targeting drug in treatment of NSCLC. EGFR-TKI can preventthe binding of ATP to ATP-binding pocket of the EGFR in a competitive manner,selectively inhibit the activity of EGFR tyrosine kinase, and then block the startup ofEGFR and its transduction pathway. By this way, the transduction pathway ofRAS/RAF/MAPK would be inactivated, the proliferation inhibited and the apoptosisinitiated of cancer cells.With the clinical generalization of the drug applications in recent years, it is foundthat there are individual differences in EGFR-TKI sensitivity among NSCLC patients.Many previous studies showed that nearly all EGFR-TKI could significantly effect onthe NSCLC harboring somatic mutations within the EGFR18,19,21exons. Moreover,these mutations are more prevalent in adenocarcinoma from Asian, female and non-smoker. About70%NSCLCs harboring EGFR activating mutations are sensitive toEGFR-TKI. Unfortunately, after continuous drug administration, more NSCLCs whichhad been sensitive to EGFR-TKI developed acquired resistance, which would letchemotherapy fail. For traditional chemotherapy, their drug resistance mechanisms havebeen studied relatively clearly. Generally, there are mainly four mechanisms reported,including the changes in drug transportation as over-expression of p-glycoprotein, thechange of the drug target as topoisomerase (Topo) expression changes in quality andquantity, the enhancement of cellular detoxification as increased expression ofglutathione transferase (GST-π) and the apoptosis inhibition as increased BCL-2expression.For EGFR-TKI, these mechanisms of traditional chemotherapy drugs cannot beadopted directly. Traditional chemotherapy drugs are usually cytotoxic andnucleus-targeting. It is necessary for the drugs to enter the cell to carry out the killing.While, EGFR-TKI acts in the cell membrane to inhibit the proliferation of tumor cells,and it does not kill tumor cells directly. Therefore, the resistance mechanism ofEGFR-TKI has become the basic research hotspot of clinical oncology recently.To the acquired drug resistance of EGFR-TKI of NSCLC, some phenomenonshave been reported as follows: secondary mutation T790M of EGFR20exon, theencoding gene mutations of KRAS, BRAF, MAPK, PI3K in the EGFR downstream ofthe signal transduction, MET gene amplification and epithelial-mesenchymal transition(EMT). Among these, the T790M mutation of EGFR20exon is the dominant reason forthe acquired resistance to EGFR-TKI in NSCLC patients.Generally, when the cells got damaged due to the traditional chemotherapy, itwould repair damaged structures automatically with its own repair system to avoid themutation accumulation and to maintain its genetic stability. It is well known that thefunction of mismatch repair (MMR) system is to repair base mismatch in the DNAreplication to maintain the stability of the cells. Thus, it is worth exploring whether thedysfunction of MMR was the major reason of T790M mutation, especially whenEGFR-TKI acted in tumor cells.In this work, we chose two cell lines NCI-H1975with T790M mutation of theEGFR and NCI-H1650without that. After long-term repeated exposure to gefitinib, thechanges of biological and mismatch repair gene expression between the two cell lineswere compared. We hope to answer how the cells with T790M mutation could get rid ofthe inhibitory effect from the EGFR-TKI. Method:1. EGFR-TKI drug resistance detection of NCI-H1975and NCI-H1650cell linesThe cell inhibition ratios in NCI-H1650and NCI-H1975cell lines were measuredby MTT. Cell apoptosis were detected by flow cytometry.2. Biological changes and the drug resistance mechanisms of NCI-H1650cell linewith EGFR exon19deletion mutation effected by EGFR-TKI2.1. Establishment of gefitinib acquired drug resistance cell line NCI-H1650/GR2.1.1. The mutation status of EGFR and KRAS of NCI-H1650cell line weredetected by polymerase chain reaction-high resolution melting analysis (PCR-HRMA)and DNA direct sequencing.2.1.2. Acquired drug resistance of NCI-H1650was induced by intermittentexposure to gefitinib with increasing concentration gradually in vitro.2.1.3. Cell inhibition ratio and drug resistance index were detected with MTT.2.1.4. The number of viable cells was calculated by Trypan blue, and then theinhibition curve of parent and drug resistance cell lines to gefitinib were plotted andcompared.2.2. The biological characteristics of NCI-H1650/GR cell line2.2.1. The cellular morphologies of NCI-H1650and NCI-H1650/GR cell lines andthe changes of cellular morphologies with the influence of gefitinib were observed andconfirmed under inverted microscope.2.2.2. The growth curve and population doubling time of parent and drugresistance cell lines were monitored by cytometry.2.2.3. The cell cycle distributions of parent and drug resistance cell lines with andwithout the influence of gefitinib were analyzed by flow cytometry.2.2.4. The cell apoptosis of parent and drug resistance cell lines with and withoutthe influence of gefitinib were analyzed by flow cytometry.2.3. Detection of some target protein expressions in EGFR signal transductionpathway of acquired drug resistance cell line NCI-H1650/GRThe protein expressions of EGFR, pY1068-EGFR, KRAS and BRAF ofparent and drug resistant cell lines were analyzed by immunocytochemistry andWestern-Blotting.2.4. Detection of EMT related protein expressions of NCI-H1650/GR cell lineThe protein expressions of E-cadherin and vimentin were detected byimmunocytochemistry. 2.5. Detection of EGFR and KRAS gene mutation in NCI-H1650/GR cell lineThe mutation status of EGFR and KRAS genes of acquired drug resistance cell lineNCI-H1650/GR were detected by PCR-HRMA.3. Biological changes and drug resistance mechanisms of NCI-H1975cell line withEGFR T790M mutation of NSCLC effected by EGFR-TKIThe methods are the same as step2.4. The expression changes of the mismatch repair protein hMSH2and hMLH1ofNCI-H1650and NCI-H1975cell lines effected by EGFR-TKI4.1. The mismatch repair protein expressions of hMSH2and hMLH1inNCI-H1650, NCI-H1975, NCI-H1650/GR and NCI-H1975/GR cell lines were detectedby immunocytochemistry and Western-Blotting.4.2. The mRNA expressions of mismatch repair gene hMSH2of NCI-H1975andNCI-H1975/GR cell lines were detected by Real-Time PCR.Results:1. EGFR-TKI drug resistances of NCI-H1975and NCI-H1650cell linesThe50%inhibitory concentration (IC50) of NCI-H1650was18.31μmol/L, whichwas higher than IC50(6.145μmol/L) of NCI-H1975. The apoptosis analysis showed thatthe apoptosis proportion of NCI-H1650was increasing in dose-dependently manner,while the proportion of NCI-H1975was not increasing dose-dependently.2. Biological changes and resistance mechanisms of NCI-H1650cell line withEGFR exon19deletion mutation of NSCLC effected by EGFR-TKI2.1. Establishment of gefitinib acquired drug resistance cell line NCI-H1650/GR2.1.1. The results of HRMA detection showed that there were EGFR19and20exon mutations in NCI-H1650cell line. DNA sequencing showed that the EGFR19exon mutation was a deletion mutation, known as delE746-A750. The EGFR20exonmutation was a nonsense mutation, known as a base2607G→A.2.1.2. The drug resistant cell line NCI-H1650/GR was obtained over12monthsof gefitinib resistance induction.2.1.3. The IC50of NCI-H1650and NCI-H1650/GR cell lines were18.31μmol/Land39.494μmol/L respectively. The resistance index of NCI-H1650/GR cell line was2.157.2.1.4. There was a negative correlation of the number of viable cells (NCI-H1650and NCI-H1650/GR cell lines) to gefitinib concentration in the same effect time. The NCI-H1650in the same gefitinib concentration.2.2. The biological characteristics of NCI-H1650/GR cell line2.2.1. The obvious morphological differences of NCI-H1650/GR cell line werefound from NCI-H1650. Most of NCI-H1650/GR cells became spindle from round orpolygonal, and the cell became bigger.2.2.2. The population doubling time of NCI-H1650and NCI-H1650/GR cell lineswere56.183±2.459h and69.414±2.067h respectively (P <0.05). The time ofNCI-H1650/GR was13.231±4.518h longer than that of NCI-H1650.2.2.3. The cell proportion of NCI-H1650/GR was increased in phase G0/G1,decreased in phase S, and the heteroploid cells were increased obviously compared withNCI-H1650. The cells of NCI-H1650cell line effected by gefitinib for24h wereinhibited in phase G0/G1. However, the cells of NCI-H1650/GR were not inhibited inphase G0/G1, but there were a lot of heteroploid cells.2.2.4. The apoptosis proportion of NCI-H1650/GR was higher than that ofNCI-H1650, but there was no significant difference (P>0.05). The apoptosisproportion of NCI-H1650was increased dose-dependently following gefitinib effectwith low concentration, but that of NCI-H1650/GR was not. The apoptosis proportionsof NCI-H1650and NCI-H1650/GR were both obviously increased following gefitinibeffect with high concentration but the proportion of NCI-H1650/GR was still lower thanthat of NCI-H1650.2.3. The expressions of some target proteins in EGFR signal transduction pathwayof acquired drug resistance cell line NCI-H1650/GRThe EGFR, pY1068-EGFR, KRAS protein expressions of NCI-H1650/GR werehigher than those of NCI-H1650, but the protein expression of BRAF was lower thanthat of NCI-H1650.2.4. The EMT of acquired drug resistance cell line NCI-H1650/GRThe E-cadherin protein expression of NCI-H1650/GR was lower than that ofNCI-H1650, but the protein expression of vimentin was higher than that of NCI-H1650.2.5. EGFR and KRAS gene mutation in NCI-H1650/GR cell lineNo new mutations were found in EGFR and KRAS of NCI-H1650/GR withPCR-HRMA.3. Biological changes and drug resistance mechanisms of NCI-H1975cell line withEGFR T790M mutation of NSCLC effected by EGFR-TKI3.1. Establishment of gefitinib acquired drug resistance cell line NCI-H1975/GR 3.1.1. EGFR20and21exon mutations in NCI-H1975cell line were confirmed byPCR-HRMA. DNA sequencing showed that the EGFR20exon mutations were T790Mand a heterozygous mutation2607G→A. The EGFR21exon mutation was L858R.3.1.2. The drug resistant cell line NCI-H1975/GR was obtained over8months ofgefitinib resistance induction.3.1.3. The IC50of NCI-H1975and NCI-H1975/GR cell lines were6.145μmol/Land12.343μmol/L, respectively. The resistance index of NCI-H1975/GR cell line was2.009.3.1.4. The negative correlations of viable cell numbers in NCI-H1975andNCI-H1975/GR to gefitinib concentration in the same exposure time were observed.The number of viable cells in NCI-H1975cell line was decreased with the timeextension of gefitinib induction, but the viable cell numbers in NCI-H1975/GR cell linewas decreased in the first day and then increased slowly with the same concentration.3.2. The biological characteristics of NCI-H1975/GR cell line3.2.1. The morphology of NCI-H1975/GR cell line was significantly different fromthat of NCI-H1975. Most of cells from NCI-H1975became spindle from long spindle,and the cells became smaller.3.2.2. The population doubling time of NCI-H1975and NCI-H1975/GR cell lineswere30.531±1.823h and46.535±0.428h respectively (P <0.05). The time ofNCI-H1975/GR was16.004±1.426h longer than that of NCI-H1975.3.2.3. The cell proportion of NCI-H1975/GR was slightly increased in phaseG0/G1, and the heteroploid cells were also increased, but there was no significantdifference (P>0.05). The cell proportions of NCI-H1975and NCI-H1975/GR cell lineswere both obviously increased in phase G0/G1and decreased in phase S followinggefitinib effect for24h. In addition, there were no heteroploid cells in the same time.3.2.4. The apoptosis proportion of NCI-H1975/GR was higher than that ofNCI-H1975(P <0.05). The apoptosis proportion of NCI-H1975/GR was decreasedfollowing gefitinib effect with low concentration (20μmol/L),(P <0.05), but theapoptosis proportion of NCI-H1975/GR were increased with high concentration (40and80μmol/L),(P <0.05). Nevertheless, the apoptosis proportion of NCI-H1975/GR waslower than that of NCI-H1975. The apoptosis proportions of both NCI-H1975andNCI-H1975/GR increased to the highest at the40μmol/L of gefitinib, but theproportions decrease when the gefitinib concentration was over40μmol/L.3.3. The expressions of some target proteins in EGFR signal transduction pathway of acquired drug resistance cell line NCI-H1975/GRThe EGFR, KRAS, and BRAF protein expressions of NCI-H1975/GR were lowerthan those of NCI-H1975, but the pY1068-EGFR protein expression was higher thanthat of NCI-H1975.3.4. The EMT of acquired drug resistance cell line NCI-H1975/GRThe E-cadherin protein expression of NCI-H1975/GR was lower than that ofNCI-H1975, but the protein expression of vimentin was higher than that of NCI-H1975.3.5. EGFR and KRAS gene mutations in NCI-H1975/GR cell lineNo new mutations were found in EGFR and KRAS of NCI-H1975/GR withPCR-HRMA.4. The expression changes of the mismatch repair protein hMSH2and hMLH1ofNCI-H1650and NCI-H1975cell lines effected by EGFR-TKIThe protein expressions of both hMSH2and hMLH1in NCI-H1975/GR werehigher than those in NCI-H1975. The hMSH2protein expression of NCI-H1650/GRwas higher than that of NCI-H1650, but the hMLH1protein expression ofNCI-H1650/GR was lower than that of NCI-H1650. The mRNA expression of hMSH2in resistant cell lines of NCI-H1975/GR was not significantly different from that ofparent.Conclusion:1. The acquired gefitinib resistant cell line NCI-H1650/GR with EGFR exon19mutation was established with drug resistance index of2.157, and the acquired gefitinibresistant cell line NCI-H1975/GR with EGFR T790M mutation was also establishedwith drug resistance index of2.009.2. The decreasing extent of cell proliferation rate of cell line with T790M mutationis larger than that without T790M mutation after long-term exposure of gefitinib.3. After long-term exposure of gefitinib, cells in cell line with T790M mutationwould stop at phase G0/G1of cell cycle when effected by EGFR-TKI again, but thecells without T790M mutation would not.4. Apoptosis inhibition probably was one of the mechanisms by which cell linewith EGFR exon19deletion mutation developed EGFR-TKI acquired drug resistance.5. The protein expressions of EGFR and KRAS increased, and the proteinexpression of BRAF decreased in the cell line NCI-H1650with EGFR exon19mutationin development of gefitinib acqired resistance. EGFR/KRAS/BRAF signal transductionpathway in NCI-H1975cell line with T790M mutation is blocked in the development. 6. The protein expression of hMSH2increased and the protein expression ofhMLH1decreased in the cell line of NCI-H1650with EGFR exon19mutation in thedevelopment peroid of gefitinib acqired resistance. The protein expressions of hMSH2and hMLH1increased in the cell line of NCI-H1975with T790M mutation in thedevelopment period.7. EMT probably was the common mechanism of EGFR-TKI acquired drugresistance of NSCLC cell lines with or without T790M mutation. |
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