| Background and PurposeNon-small cell lung cancer(NSCLC) is the most common cancer and the leading cause of death from cancer in China and worldwide. It has been extensively proved that ptients with NSCLC harbour sensitive mutations of the epidemal growth factor receptor(EGFR) were highly responsive to EGFR tyrosine kinase inhibitors(TKIs). However, the overwhelming majority of these patients inevitably acquired resistance to TKI drugs.Studies over the last few years have identified four different EGFR-TKIs resistance mechanisms:1. a secondary mutation in EGFR, EGFR 790M; 2. gene mutation or amplification of EGFR downstrem molecules; 3. amplification of the MET oncogene or other singnaling pathway; 4. Epithelial-Mesenchymal Transition(EMT) or translate to Small Cell Lung Cancer(SCLC).In this study, we chosed two EGFR mutated cell lines–HCC827(19 exon deletion) and NCI-H1975(L858R/T790 M mutation)–to induce TKIs drug resistance cell lines. After long-term exposure to TKIs, we got the gefitinibresistance cell line(HCC827/GR), the afatinib-resistance cell line(HCC827/AR) and AZD9291-resistance cell line(HCC827/AZDR). Whereafter,we performed several experiments and tried to find the mechanism of TKIs drug resistance from these cell lines. MethodsIn part one, in order to identify the HCC827 and NCI-H1975 cell lines, we detected the mutations of exon 19-21 of EGFR by direct DNA sequencing, then examined the growth inhibition of TKIs by MTS methods and calculated IC50 respectively.Thereafter, we developed gefitinib-resistance cell line HCC827/GR, afatinib-resistance cell line HCC827/AR and AZD9291-resistance cell line HCC827/AZDR by long-term exposure to drugs. To examine their drug tolerance, we examined the growth inhibition of drug-resistance cell lines with it’s resistant drug by MTS method, then calculated IC50 respectively. We treated HCC827 and drug-resistance cell lines by different concentrations of gefitinib, afatinib or AZD9291, then analysed the growth of the cells by plotting cell growth cruve.In part two, for exploreing mechanism of drug-resistant, we detected EGFR and proteins of EGFR downstream signaling pathway of drug-resistance cell lines. We treated HCC827 and HCC827/AR cell lines by afatinib at the concentrations of 0n M, 10 n M, 100 n M and 1000 n M for 6 hours, then collected the total proteins and detected EGFR and p Y1068-EGFR protein by Western Blot;and treated HCC827 and HCC827/AZDR cell lines by AZD9291 at the concentrations of 0n M, 10 n M, 100 n M and 1000 n M, then collected total proteins and detected EGFR and p Y1068-EGFR protein by Western Blot. Then we detected EGFR and p Y1068-EGFR protein of HCC827 and HCC827/AZDR cell lines by immunofluorescence technique. Thereafter, we detected proteins of MAPK signal by Western Blot.In order to reveal the relationship of MAPK signal and drug resistance, we made HCC827/AZDR cell lines exposed to Trametinib or SCH772984 and collected it’s proteins and detected p ERK protein of by Western Blot, then we examined the growth inhibition of HCC827/AZDR cell line at 10 n M trametinib and different concentrations of AZD9291 by MTS methods, then calculated the IC50 of AZD9291. ResultsIn part one,we choosed the HCC827 cells to induce TKIs-resistance cell lines after verified the HCC827 and NCI-H1975 cell lines. We developed the gefitinib drug-resistance cell line HCC827/GR after 9 months exposure to gefitinib(three rounds of strike and final maintained concentration 300 n M). The inhibition rate of HCC827/GR by different concentrations of gefitinib was consistently lower than parental cell line(P<0.05). However, we couldn’t calculat the IC50 by the MTS method. Cell growth showed that the growth of HCC827/GR cell line was significantly slower than parental cell line. Cell proliferation rate of HCC827/GR cultured at gefitinib 30 n M was approximate that of without gefitinib(P>0.05), while at gefitinib 300 n M or 1000 n M was significantly slower than without gefitinib(P<0.05).We developed the afatinib drug-resistance cell line HCC827/AR by 7 months exposure to afatinib(maintained concentration from 0.5 to 16 n M gradually). The IC50 of afatinib in HCC827/AR cell line was 4667 n M, and the resistance index of HCC827/AR cell line was 1866.8. Cell growth showed that there was non significantly diffrence between the growth of HCC827/AR cell line and parental cell line. Cell proliferation rate of HCC827/AR cultured at afatinib 16 n M was approximate that of without afatinib(P>0.05).We developed the AZD9291 drug-resistance cell line HCC827/GR after 6 months exposure to AZD9291(three rounds of strike and final maintained concentration 160 n M). The IC50 of AZD9291 in HCC827/AZDR cell line was 6116 n M, and the resistance index of HCC827/AR cell line was 459.8. Cell growth showed that there was non significantly diffrence between the growth of HCC827/AZDR cell line and parental cell line. Cell proliferation rate of HCC827/AZDR cultured at AZD9291 160 n M was approximate that of without AZD9291(P>0.05).In part two,the EGFR and p Y1068-EGFR proteins of HCC827/AR and HCC827/AZDR cell lines were significantly lower than parental cellline(P<0.01). The concentration of EGFR and p Y1068-EGFR proteins in HCC827, HCC827/AR and HCC827/AZDR cell lines were decreased while the concentartion of drugs increased(P<0.05). All were detected by Western Blot. The EGFR and p Y1068-EGFR proteins of HCC827/AR and HCC827/AZDR cell lines were significantly lower than parental cellline, detected by immunofluorescence technique.The p MEK 1/2 and p ERK 1/2 protein of HCC827/AR cell line was significantly less than parental cell line, and there was no concentration-response relationship between afatinib and p MEK and p ERK 1/2 protein in HCC827/AR cell line. The p MEK and p ERK 2 protein of HCC827/AZDR cell line was significantly more than parental cell line, and there was no concentration-response relationship between AZD9291 and p MEK protein in HCC827/AZDR cell line.Trametinib could reduce p ERK 1/2 protein in HCC827/AZDR cell line at the concentration of 10 n M(P<0.05), while SCH772894 could inhibite the phosphorylation of ERK 1/2 protein in HCC827/AZDR cell line at the concentration of 100 n M(P<0.05). The IC50 of AZD9291 in HCC827/AZDR cell line while dissolved into 10 n M trametinib was 6215 n M, without significant difference with the IC50 of AZD9291. Conclusion1. We finally established two stable resistant cell lines: afatinib-resistance cell line HCC827/AR and AZD9291-resistance cell line HCC827/AZDR.2.The EGFR and p Y1068-EGFR proteins of HCC827/AR and HCC827/AZDR cell lines were significantly lower than parental cellline. It’s an crucial mechanism of drug resistance in these two cell lines.3. The MAPK pathway was actived in a high lever in HCC827/AZDR cell line. Trametinib(inhibitor of MEK) could not reduce the IC50 of AZD9291 in HCC827/AZDR cell line, so the activation of a MAPK pathway was not the key mechanism of drug resistance. |
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