| Objective1.Clinical study:To observe the efficacy of elemene emulsion injection plus TKIs on patients with stage IIIB/IV NSCLC(non-small cell lung cancer)with activating EGFR mutations(exon 19 deletions or exon 21 L858R mutations)and postprogression the first generation of EGFR-TKIs therapy.In this way,we can test whether elemene emulsion injection can reverse the TKI resistance ang prolong the survival time.Thus it can probably offer a new theraputic choice for TKI resistant patients beyond progession.2.Cell experiment:Testing the cell proliferation,cell cycle,c-met mRNA and p-met,met,EZH2,p-Akt,Akt proteins expression of HCC827 cells and A549 cells in order to study the signal path related to the TKI resistance machanism in which elemene emulsion injection perform its antitumor activity,hoping that it would make up the deficiency of targeted therapy in a TCM way.Methods1.Clinical study:This is a single-arm study.The enrolled patients include stage IIIB/IV NSCLC patients with EGFR positive mutations(exon 19 deletions or exon 21 L858R mutations)and postprogression the first generation of EGFR-TKIs therapy(Gefitinib,Erlotinib or Icltinib).The clinical progression model must be slow progression(the tumor enlarge slightly and the symptom score is no more than 1).The enrolled patients were treated with continuing TKI in the original concentration(gefitinib 250mg/day or erlotinib 150mg/day or Icotinib 125mg/day)plus elemene emulsion injection which is dissolved in 400ml of glucose injection.The elemene emulsion injection was taken once a day for 14 days,then rest for 14 days,and repeated every 28 days.The blood routine examination,hepatorenal function and tumor makers were tested before and after every cycle of treatment.The imageological examination was taken every 2 cycles of elemene emulsion injection.The changement of the patients' symptom and other related datas were collected for analyse.When the imageological examination indicated tumor progression again and the clinicians determined the necessity of changing the theraputic strategy,the study was finished and the median PFS,living quality and safety were evaluated.2.Cell experiment:The HCC827 cells and A549 cells were used for experiment.The cells were intervened with elemene emulsion injection in different doses.We used MTT assay to test the cells proliferation,flow cytometry to test the cells cycle,western blot to test the expression of the p-met,met,EZH2,p-Akt,Akt proteins,the real-time PCR to test the c-met mRNA expression.Results1.Clinical study:(1)Twenty patients were enrolled in this study,in which twenty patients were included for ITT analysis and nineteen patients were included for PPA analysis.Median PFS was 3.1(95%CI:2.60,3.54)months.Dividually,median PFS in patients with EGFR exon 19 deletions and exon 21 L858R mutation was 3.07(95%CI:1.50,4.65)months and 2.67(95%CI:1.74,3.60)months.In these 20 patients,13 patients were treated with gefitinib,their PFS was 2.83 months(95%CI:2.33,3.34);5 patients were treated with erlotinib,their PFS was 2(95%CI:0.85,3.15)months;2 patients were treated with icotinib,because of the deficient number of cases,these two patients could not meet the requirement of survival analysis.Eighteen patients reached the end point of observation,in which 1 patient died,1 patient had both an increase in the size of target lession and new lessions at PD,the other 17 patients had new lessions at PD.(2)Compared to the living quality before the conbination of elemene emulsion injection,the living quality during the treatment of TKI plus elemene emulsion injection was not worse(P>0.05).(3)Among the twenty treated patients,only one patient suffer an adverse event of phlebitis at grade 1,all the other nineteen patients didn' t have any adverse events.2.Cell experiment:(1)The MTT assay indicated the elemene emulsion injection inhibit the proliferation of HCC827 cells and A549 cells in a time-dose-dependent manner.When the doses ranged from 20 to 180 ? g/ml,in the same time,the higher doses,the greater inhibition of elemene emulsion injection on HCC827 cells proliferation,and had significant difference between groups(P<0.05).When the doses ranged from 60 to 160 ? g/ml,the longer time,the greater inhibition of elemene emulsion injection on HCC827 cells proliferation,and had significant difference between 24 hours and 48 hours(P<0.05),but had no significant difference between 48 hours and 72 hours(P>0.05).Similarly,when the doses ranged from 20 to 180? g/ml,in the same time,the higher doses,the greater inhibition of elemene emulsion injection on A549 cells proliferation,and had significant difference between groups(P<0.05).when the doses ranged from 40 to 140 ? g/ml,the longer time,the greater inhibition of elemene emulsion injection on A549 cells proliferation,and had significant difference between groups(P<0.05).(2)The flow cytometry result indicated that the HCC827 cells and A549 cells were mainly blocked in phase G0/G1 of the cell cycles by elemene emulsion injection.(3)The real-time qPCR indicated that the elemene emulsion injection can decrease the c-met mRNA.As the dose of elemene emulsion injection increased,the expression of the c-met mRNA reduced,and there were significant difference between the intervened groups and the control group(P<0.05).(4)The western blot result indicated that the elemene emulsion injection can downregulate the the expression of p-met?met?EZH2 and p-Akt protein in both HCC827 cells and A549 cells.Conclusion(1)Continuing TKI plus elemene emulsion injection beyond slow progression is feasible in stage IIIB/IV NSCLC patients with EGFR mutation-positive and resistant to the first generation of EGFR-TKIs.The median PFS was 3.1 months,with a good living quality and safety.(2)Elemene emulsion injection can significantly inhibite the proliferation of HCC827 cells and A549 cells in vivo experiment.It mainly blocked the cells in phase G0/G1 of the cell cycles.On the level of proteins and genes,elemene emulsion injection may perform its anti-tumor function by downregulating the expressions of c-met mRNA and p-met met,EZH2,p-Akt proteinS. |
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