| Objective: Lung cancer is a kind of malignant tumor threatening humanhealth seriously, its incidence and mortality have been increasing year by yearin China, of which non-small cell lung cancer (NSCLC) accounts for morethan80%. Although the technological level of surgery, chemotherapy andradiotherapy is continuously improving, their efficacy in lung cancer is stillunsatisfactory. In recent years, the epidermal growth factor receptor tyrosinekinase inhibitor (EGFR-TKI), gefitinib and erlotinib, have been used toclinical treatment for NSCLC, which is considered to improve the survivalrate of some patients. However, the primary and acquired resistance toEGFR-TKI of NSCLC patients limits its clinical application, so we mustexplore new strategy or method to overcome this problem. Recently, someforeign literatures indicated that as anti-tumor drug of multiple targets, heatshock protein90inhibitor can be used to treat NSCLC, but for differentNSCLC cell lines being primary and acquired resistance to EGFR-TKI, theconclusion of whether or not they all have antitumor activity were not beenunified. As found to be overexpressed in many malignant tumors, HSP90played an important role in cell proliferation, differentiation, invasion andmetastasis. Its client proteins include various signal transduction factors, soinhibiting HSP90can play antitumor effect by a variety of pathways and links,this provided new idea for resolving resistance of TKI. Currently, manyclinical trials of HSP90inhibitors for treating malignant tumors have beenperformed and satisfactory results have been obtained. As a kind of HSP90inhibitor,17-DMAG is the derivative of geldanamycin, it has better watersoluble and higher oral bioavailability than17-AAG and the phase I clinicaltrials of17-DMAG was already accomplished in a wide range of tumors. Inthis study, we examined the effect of17-DMAG on NSCLC cell lines A549 and H1975which were primary and acquired resistant to EGFR-TKIrespectively, the purpose was to explore its influence on cell proliferation,apoptosis and the expression of EGFR in vitro as well as their possiblemechanism.Methods: We treated non-small cell lung cancer cell lines A549andH1975with different concentrations of17-DMAG respectively. The inhibitionof cell proliferation was measured by MTT assay for24h,48h and72h. Flowcytometry with PI simple staining was used to analyze the cell apoptosis andwestern blot was adopted to detect the expression of HSP90and EGFR after17-DMAG treated for48h.Results:1MTT assay showed that after exposed to0.003,0.03,0.3,3,30μmol/Lof17-DMAG in A549cell line, the inhibitory rate were10.51±1.65%,17.60±1.43%,32.32±2.72%,42.58±3.31%,54.49±0.95%for24h,21.89±1.57%,28.76±3.06%,38.45±1.74%,57.38±1.21%,73.73±2.42%for48h and29.15±1.19%,38.64±2.31%,60.23±1.26%,78.45±1.34%,88.90±0.89%for72h respectively; in H1975cell line, the inhibitory rate were8.75±2.43%,13.50±1.54%,22.71±3.54%,45.21±3.84%,70.51±2.41%for24h,16.43±2.63%,28.22±2.19%,40.30±5.23%,59.82±2.05%,81.17±5.38%for48h and28.81±3.06%,39.68±1.05%,56.26±2.50%,75.33±3.08%,90.61±2.51%for72h respectively.17-DMAG inhibited the cell proliferationof A549and H1975in different concentrations and time, the inhibitory rate ofall groups were significantly higher than control group (P＜0.01), and all theeffects were in time-and dose-dependent manner.2The results of flow cytometry were that the apoptosis rate of A549andH1975cell lines in the control group were0.77±0.13%and1.52±0.17%respectively; after exposed to0.003,0.03,0.3,3μmol/L of17-DMAG for48h,the apoptosis rate of A549cell line were2.13±0.21%,3.32±0.23%,6.43±0.40%,11.73±0.26%, and the apoptosis rate of H1975cell line were3.19±0.19%,5.95±0.21%,9.83±0.35%,16.88±0.14%respectively. Bypairwise comparison with SNK of ONE-WAY ANOVA, we found that the apoptosis rate of all drug groups were significantly higher than control group(P＜0.01), and the effects were in dose-dependent manner.3By western blot analysis we detected that in A549cell line the ratios ofEGFR/GADPH and HSP90/GADPH in the control group were0.261±0.070and0.128±0.007respectively; after exposed to0.003,0.03,0.3,3,30μmol/Lof17-DMAG for48h, the ratios of EGFR/GADPH were0.249±0.069,0.259±0.090,0.329±0.075,0.228±0.056,0.219±0.030, and the ratios ofHSP90/GADPH were0.136±0.008,0.122±0.006,0.153±0.034,0.128±0.015,0.118±0.034respectively. The difference among the expression of both HSP90and EGFR protein in all drug groups and control group was not significant (P＞0.05); in H1975cell line the ratios of EGFR/GADPH and HSP90/GADPHin the control group were1.397±0.005and0.107±0.027respectively; afterexposed to0.003,0.03,0.3,3,30μmol/L of17-DMAG for48h, the ratios ofEGFR/GADPH were0.978±0.013,0.706±0.007,0.303±0.006,0.101±0.012,0.018±0.003, the ratios of HSP90/GADPH were0.108±0.021,0.100±0.024,0.105±0.024,0.102±0.023,0.093±0.019respectively. The difference amongthe expression of HSP90protein in all drug groups and control group was notsignificant (P＞0.05), but the expression of EGFR protein in all drug groupsand control group was statistically different (P＜0.01). This result suggestedthat17-DMAG can suppress the expression of EGFR protein in H1975cellline and the effect was in a dose-dependent manner.Conclusions: HSP90inhibitor17-DMAG inhibited the proliferation ofNSCLC cell lines A549and H1975being primary and acquired resistant toEGFR-TKI respectively, and the effect was in time-and dose-dependentmanner, it also induced apoptosis of both cell lines.17-DMAGdown-regulated the expression of mutant EGFR protein with the increasing ofdrugˊs concentration while this phenomenon was not observed inEGFR-wild type cell line, this suggested that its mechanism was differentbetween A549and H1975cell lines with different genetic backgrouds. Our study provided new idea for treatment of NSCLC being resistant toEGFR-TKI. |
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