| Background and objective It is well known that lung cancer is the leading cancer killer,and the incidence rate continues to increase.According to the pathological features of lung cancer were divided into non small cell lung cancer and small cell lung cancer,but according to the epidemiological survey found that NSCL caccounted for 80% ~ 85%.About 70%-80% of patients are in the middle and late stages of diagnosis and can not be treated with surgery.Therefore,non-surgical treatment plays an important role in most lung cancer patients.Currently,cytotoxic chemotherapy is an effective treatment option for this population,but the total survival time(OS)is about 12 months,and the 3 and 5year survival rates of patients are only 19% and 11% respectively.Although radiotherapy and chemotherapy and targeted therapy are the main targets for the treatment of advanced non-small cell lung cancer,the effective rates of radiotherapy and chemotherapy are only between 20% and 30%.And reversible inhibition of TKI targeted drug EGFR,drug use in about 10 months after the emergence of drug resistance,even deterioration of the disease.All of the Erb B receptor family of second generation TKI drugs irreversibly inhibits afatinib,although the growth of cancer cells by more comprehensive,more durable,but also the inevitable resistance.How to develop a more economical and effective treatment mode is one of the current medical research urgent matters.Ethacrynic acid(EA)is a kind of electrophilic diuretic,is the first to be used as antitumor drugs glutathione transferase S(GSTs)inhibitors,the substrate can be directly applied to GSTs to block its function,or glutathione and glutathione(GSH)thiol michael addition reaction to remove the body of GSH,inhibiting the activity of GSTs and enhancing the sensitivity of tumor cells.Recent studies have found that EA and its derivatives can act as inhibitors of GST and WNT in addition to the diuretic effect,and can exert a certain antitumor effect in breast cancer.Even recent studies have found that reducing glutathione synthesis in vivo can reverse the resistance to imatinib,an irreversible TKI inhibitor.About the use of afatinib with ethacrynic acid in lung cancer has not been reported,so we plan to study a method for the treatment of imatinib combined with EA mutations in lung cancer,to analyze the effect of the combination on the one hand,on the other hand,to explore its function mechanism,to provide theoretical and experimental basis for non drug combination small cell lung cancer.Methods We studied the effects of the combination of afatinib and EA in A549 and H1975.The effects of afatinib and EA on A549 and H1975 cells proliferation were determined by CCK8 assay.Annexin V-FITC and PI double staining was used to analyze apoptosis.To investigate Doxycycline on human small cell lung cancer cells,GSTs levels in the cell were detected by ELISA.The abilities of metastasis and invasion in lung cancer lines were detected by Transwell.We further studied the mechanism of synergy.The gene's levels(EGFR?P53?TNFSF14?VEGFA?WNT5A?WNT10A?MAPKAP1 and MAPK8IP3)of afatinib and EA on H1975 cells were determined by RT-PCR.The secondary resistance of H1975 as the research object,the control group,single drug group and combined treatment group after 48 h,for transcriptome sequencing,gene expression differences among the groups,clustering analysis and GO/Pathway enrichment analysis.To establish a xenograft animal model,human lung cancer A549 and H1975 cells were injected into the BALB/c nude mice subcutaneously.Treatment with afatinib and/or EA inhibited the growth of human NSCLC A549 and H1975 cells xenografts.Results1.1 The inhibition effect of afatinib combined with EA on the growth of A549 and H1975 cells We obsersved that afatinib or/and EA had differentence for inhibition of cell proliferation effects with the treatment of 48 h.With the increasement of drug concentration,the inhibitory effect gradually inereased.Antiproliferative effects of combination group was stronger than any regimen alone on equal concentration or higher concentration,strengthen synergetic effect of anti-tumor.One-way ANOVA test analysis showed that obviously antiproliferative effects of combination group was stronger than afatinib group alone,the inhibition rate increased signifieantly and appeared dose-dependment effect,the difference was significant(p<0.05).It indicated that co-treatment with afatinib and EA has synergetic antiproliferative effect for NSCLC drug-resistance cells.CDI values of combined-drugs were calculated by Bio Calcusyn software,the result showedthat the more drug concentration inereased,the more inhlbitory effect inereased,,indieating that a combination of both afatinib and EA has a synergistic inhibition effect on NSCLC cells.It showed a strong synergistic effect(Cl<0.3)of H1975 cells.1.2Afatinib combined with EA could inhibit the increase of A549 and H1975 cells in vitro and in vivo CCK8 method was used to detect the effect of the same concentration on the proliferation of A549 and H1975 cells.The results showed that the rate of cell inhibition in the combination group was significantly higher than that in group afatinib and group EA at the same concentration.As shown in Figure 6,the effect of H1975 on inhibiting proliferation was more obvious than that of A549.The growth effect of implanted tumor by combination with afatinib and EA on nude mouse xenograf tumor model Tumor volume was measured at the end of experiment.The average tumor volumes of afatinib,EA,afatinib and EA group were lower than the control group.The tumor volumes were respectively(1554.7± 340.22)mm3,(604.6 ± 87.80)mm3,(1093.5 ± 144.53)mm3,(402.8 ±89.61)mm3;(870.7±126.23)mm3,(484.1±76.65)mm3,(723.3±135.72)mm3,(82.9 ± 24.71)mm3.Volume of combined group has signifieantly lowered(P=0.000);tumor inhibitory rates were 0.00%,51.75±9.17%,29.25±10.37%,69.76±4.25%;0.00%,48.72±10.02,23.54±12.76,84.12±8.27 respectively,combination group was the best,F=18.345 ? 50.192.It indicated that afatinib combined EA can synergistically inbibit tumor growth which wasconsistent with the first part of experiments.apoptosis of A549 and H1975CCK8 experiments show that compared to treatment of single drug,combination therapy with afatinib and EA can synergistically inhibit cell growth of A549 and H1975 which may be related to cell cycle and apoptosis and its mechanism,so the effect of tumor cell apoptosis of afatinib or/and EA was detected by flow cytometry.The results of cell cycle showed that compared to treatment of control group and single drug groups,combination therapy group can synergistically arrested G0/G1 phase of cell cycle in A549(p<0.05)and S+G2 phase in H1975(p<0.05).The results of cell apoptosis showed that afatinib or EA treatment alone in A549 and H1975 cells for 48 hours can induce cell apoptosis,apoptosis rates were 2.5%?2.9% ?11.3%?4.7% respectively.However,after combination treatment,apoptosis significantly inereased in A549?H1975 cells,was 5.7% ?41.8%(p<0.05),have significant difference.It indieated that co-reatment possesses synergistic effect of promoting apoptosis especially for H1975.1.4 Combination with afatinib and EA synergistically effect cell abilities of invasion and metastasis of A549 and H1975 Transwell experiments show that compared to treatment of control group and single drug group.The results shows that single drug group,combination therapy with afatinib and EA group through the cell membrane into the lower chamber than the control group,cell numbers of therapy with afatinib and EA group through the cell membrane into the lower chamber were decreased,the differences were statistically significant(p<0.05).In the invasion assay,cell numbers of control group,single drug group and herapy with afatinib and EA group through the cell membrane into the lower chamber were decreased,the differences were statistically significant(p < 0.05).And the A549 cell,the1.3 Combination with afatinib and EA synergistically effect cell cycle andnumbers of cell through the cell membrane into the lower chamber didn't see significant decrease,there was no statistically significant difference(p<0.05).1.5 Study on the detection and menchanism of transcriotome Combination with afatinib and EA of H1975 Transcriptome analysis is the study of gene expression at RNA level.Transcriptome sequencing(RNA sequencing)can be used to study gene function and gene structure,which can reveal the biological process of drug target.The secondary resistance H1975 as the object in this study,the control group,single drug group and combined treatment group after 48 h,then RNA was extracted for transcriptome sequencing.Trough the analysis,we found differential gene expression of EGFR and P53,we find TNFSF14,VEGFA,WNT5 A,WNT10A,MAPKAP1 and MAPK8IP3 related gene.The results by RT-PCR are basically consistent with the sequencing results indicated that the transcription group side learning results are reliable.Through the analysis we found the differentially expressed genes found mainly with 20 pathways,which is most closely associated with cancer Pathway in cancer,Wnt signaling pathway and cytokine-cytokine receptor interaction of the three signal pathways.Based on the analysis of gene expression differences,we found 155 up-regulated and 175 down regulated genes.(filter: FDR < 0.01,Log = 2 and Fold-change =-2,Pvalue < 0.001).To provide important data resources for further study on the molecular mechanism of drug resistance to NSCLC.Conclusions:1.Afatinib combined with EA has synergistic antiproliferation effect on A549 and H1975 human lung cancer cells.CI values of combined-drugs werecaleulated by Chou-Talalay software,the synergistic effect of A549 on the proliferation was significant at the higher dose(afatinib IC50+EA IC50),while the proliferation of H1975 was stongly inhibited,and the CI I<0.3.2.Afatinib combined with EA has inhibitory tumor growth effect on non small cell lung cancer on A549 and H1975 in vitro and in vivo,especially for the secondary drug resistant lung cancer of H1975.3.Afatinib,EA and afatinib combined with EA can inhibit the cell cycle of A549 and H1975,and can synergistic promoting-apoptosis effeets on A549 and H1975 human lung cancer cells,and can inhibit the migration and invasion of A549 and H1975 cells.4.Afatinib combined with EA has significantly synergistic inhibitory effects on phosphorylation levels of EGFR,P53,TNFSF14,VEGFA,WNT5 A,WNT10A,MAPKAP1 and MAPK8IP3 on H1975 human lung cancer cells;and may be possible to play the role of secondary resistance NSCLC cells in some of these three pathways(Pathway in cancer ? Wnt signaling pathway and cytokine-cytokine receptor interaction. |
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