| Objective: Lung cancer is a malignant tumor with high morbidity rate and mortality rate,diffrent pathological types have diffrent biological behaviour.The pathological characteristics include high malignent degree,easy to recurrence and metastasis and the 5-year survival rate is low.The leading cause of death and the failure of clinical treatment in patients with advanced lung cancer is distance metastasis.In recent years,molecular targeted drugs in the treatment of non-small cell lung cancer has achieved breakthrough progress.The epidermal growth factor receptor-tyrosine kinase inhibitors are the most representative drug which it has the characteristics of high efficientcy,low toxicity and it was recommended as first choice of non-small cell lung cancer with EGFR mutations.The main mechanism is suppressing the activation of tyrosine kinase structural domain,blocking the conduction of signal pathway and playing an important role in inhibiting metastasis of tumor cells and promoting apoptosis of tumor cells.However,clinical observation found that the part of the patients of taking erlotinib not only the tumor growth is suppressed,but also the tumor metastasis is effected.So,erlotinib may regulation the tumor microenvironment by a potential way,and then affect tumor metastasis,but the specific mechanism is still remains unclear.The adhesion molecules CD44 are an important member in adhesion molecules family in tumor microenvironment,it cans mediated the adhesion role between cells and cells,cells and matrix.Further,it cans provide enough power for tumor metastasis through take participating in a series of signal transduction.Between EGFR signal pathway and CD44 may be has some kind of cross collaborative relationship in the research results of head and neck cancer and breast cancer.But it is unclear in non-small cell lung cancer,and the affect is unknown for tumor metastasis.Therefore,in this study,we willdiscuss erlotinib inhibition tumor metastasis whether there is associated with down-regulation expression of CD44 by a series of experiments in vitro in NSCLC cell lines with EGFR mutations,and then the initial explore of the key signal molecules between EGFR signal pathway and CD44.This will seek more new drug targets for tumor metastasis and provide more basis evidence for drug combination.Methods:1 Cell culcure: HCC827 and A549 cells were cultured in RPMI1640 medium,15% heat-inactivated fetal bovine serum,penicillin 100 U/m L,streptomycin 100 μg/m L at 37℃ in an incubator,containing 5% CO2.2 Real-time unmarked cell proliferation experiment detect the sensitivity of erlotinib for HCC827 and A549 cells,and determine the cells of the experiment.3 The anti-proliferative effect of different concentration erlotinib for HCC827 cells were detected by MTT and the drug treatment time is 48 h.Then,calculating the proliferation inhibiting rate and IC50.4 The invision and migration ability of HCC827 cells were measured by Transwell invision assay and wound healing assay.Methods to set up control group,EGF stimulation group,erlotinib + EGF experimental group and CD44 neutralizing antibodys + EGF experimental group.5 The cell expression level of CD44 in three groups include control group,EGF stimulation group,erlotinib + EGF treatment group were detected by Flow cytometry.6 The protein expression level of CD44 in three groups include control,EGF stimulation group,erlotinib + EGF treatment group were detected by western blotting.7 The gene expression level of CD44 in three groups include control,EGF stimulation group,erlotinib+EGF treatment group were detected by RT-PCR.8 Western blotting assay is applied to detect the protein expression level of CD44,STAT3.P-STAT3 in control group,EGF stimulation group,erlotinib+ EGF experimental group and stat3 blocking agent + EGF experimental group.9 Statistic analysesThe results were expressed as mean ± standard deviation or median ±quarterback range and evaluated with SPSS 21.0 solftware by analysis of variance(One-Way ANOVA)or Kruscal-Wallis Test.Fisher’s Least Significant Difference(LSD)was used between two groups compared.P<0.05 was considered statistically significant.Results:1 Real-time unmarked cell proliferation experiment(Fig.1): the proliferat-ion inhibition effect of erlotinib was more stonger and presented a time-dependent in HCC827 cells,but the situation is not exist in A549 cells.So we chose the HCC827 cells as experiment cells.2 MTT test(Fig.2,Table1): The result show that the cell proliferation inhibition rate is gradually increasing in different concentrations of erlotinib(0.001,0.01,0.1,0.5,1,10μM)treatment the HCC827 cells for 48 h and presented a dose-dependent.The difference was statistically significant(P<0.05).IC50= 0.323μM.3 Transwell chamber invasion assay(Fig.3,Fig.4,Table2): The numbers of across membrane of 24 h were(64.07±1.51),(129.53±4.20),(21.0±1.06)and(23.87±1.70)in control,EGF stimulation group,erlotinib + EGF treatment group and CD44 neutralizing antibodys + EGF treatment group,respectively;Wound healing assay(Fig.5,Fig.6,Table3): The migration distance of 24 h were(78.65±3.19)μm,(119.98±1.62)μm,(51.73±4.23)μm,(53.18±6.71)μm in different groups,respectively.The invision and migration ability were weake-ned by erlotinib and this may be associated with down-regulation expression of CD44(P<0.05).4 Flow Cytometry(Fig.7,Table4): The cell expression level of CD44were(25.87±3.46)%,(48.37±2.21)%,(15.50±1.11)% in control,EGF stimulation group,erlotinib + EGF treatment group,respectively.The difference was statistically significant(P<0.05).5 Western blotting assay(Fig.8,Table5): The ratios of IOD between CD44 and GAPDH protein were(0.72±0.03),(0.83±0.04),(0.21±0.03)in control,EGF stimulation group,erlotinib+EGF treatment group,respectively.The difference was statistically significant(P<0.05).6 q RT-PCR assay(Fig.9,Table6): Assuming that the control group was 1with expression of CD44 m RNA,EGF stimulation group is(2.22±0.17)times as much as in the control group;erlotinib+EGF treatment group is(0.50±0.04)times as much as in the control group;Above all,the results showed that the expression of CD44 was down-regulated after erlotinib blocked the EGFR signaling pathway(P<0.05).7 Western blotting detect the protein expression of CD44,p-STAT3 and T-STAT3 in control,EGF stimulation group,erlotinib + EGF treatment group and STAT3 blocking agent + EGF treatment group,respectively(Fig.10,Fig.11,Fig.12,Table7);Our finding show that erlotinib could significantly decrease the expression of CD44 and p-STAT3 compared with control group in HCC827 cells.In addition,the STAT3 specific blocking agent could also remarkably inhibit the CD44 expression while blocking the EGFR/STAT3 signaling pathway.Above all,we found that the effect is possibly due to suppress the conduction of STAT3 signaling pathway(P<0.05).But the mechanism should be further defined.Conclusion:1 HCC827 cells are highly sensitive to EGFR-TKI and more suitable for the experiment.2 EGFR-TKIs can also significently inhibit cell metastasis while surppress the proliferation of HCC827 and it mays be related with the expression of CD44 was down-regulated.3 EGFR-TKIs can remarkably suppress the expression of CD44,and then the STAT3 signaling pathway may playing an important role in this process. |
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