A Research On Antitumor Activities And Molecular Mechanisms Of EGFR-TKIs In Esophageal Squamous Cell Carcinoma

Objective:The purpose of this study is to investigate the antitumor activities and molecular mechanisms of EGFR-TKIs?epidermal growth factor receptor-tyrosine kinase inhibitors?in esophageal squamous cell carcinoma?ESCC?cell lines and to provide novel ideas for the comprehensive treatment of ESCC.Methods:In this study,CCK-8 assays were performed to detect the antitumor activity of molecular targeted drugs on esophageal squamous cell carcinoma in vitro.Xenografts of SPF grade BALB/ca-nu mice with ESCC cell lines by subcutaneous tumorigenesis were established to discover the antitumor activity of molecular targeted drugs on ESCC cells in vivo.Western-blotting experiments were performed to find the alternation of expressions of proteins and signaling pathways in ESCC cell lines treated with molecular targeted drugs.Flow Cytometry was used to detect the changes of the cell cycle and apoptosis of ESCC cell lines treated with molecular targeted drugs.Results:In vitro:In KYSE30 ESCC cell line,the IC₅₀ value of afatinib and erlotinib were 0.04615?95%confidence intervals 0.01240 to 0.1718,r²=0.8218?and 0.1500?95%confidence intervals 0.07653 to 0.2939,r²=0.9473?,respectively.In KYSE150ESCC cell line,the IC₅₀ value of afatinib and erlotinib were 0.04089?95%confidence intervals 0.02331 to 0.07174,r²=0.9645?and 0.4085?95%confidence intervals0.1812 to 0.9207,r²=0.9254?,respectively.In KYSE450 ESCC cell line,the IC₅₀ of afatinib and erlotinib were 56.84?95%confidence intervals 6.634 to 507.7,r²=0.8645?and 10.69?95%confidence intervals 0.8571 to 133.3,r²=0.6724?,respectively.In KYSE510 ESCC cell line,the IC₅₀ value of afatinib and erlotinib were 3.616?95%confidence intervals 0.4716 to 27.72,r²=0.6229?and 40.64?95%confidence intervals18.18 to 90.85,r²=0.9661?,respectively.In EC109 ESCC cell line,the IC₅₀ of afatinib and erlotinib were 0.01330?95%confidence intervals 0.01016 to 0.01740,r²=0.9904?and 0.02394?95%confidence intervals 0.01691 to 0.03388,r²=0.9851?,respectively.In vivo:The subcutaneous tumor weights of afatinib group were significantly lower than erlotinib group?P<0.0001?and vehicle group?P=0.0002?,the differences were statistically significant.However,the difference of subcutaneous tumor weights between erlotinib group and vehicle group was not statistically significant?P=0.5524?.It can also be inferred by one-way ANOVA that the differences of subcutaneous tumor weights between the three groups were statistically significant?P<0.0001?.Comparing with erlotinib group and vehicle group,the weights of nude mice in afatinib groups had a rapid decline in the first three days?P=0.0185?,and the trend slowed down during the 4^th to 6^th days?P=0.0006?.After six days the weights of afatinib group began relaxedly picking up and ultimately reached similar with erlotinib group and vehicle group in the terminal record?P=0.812?.Western-blotting:In KYSE30 cell line treated with afatinib,the expression levels of pEGFR and pERK were significantly down-regulated compared with untreated KYSE30 ESCC cell line.The expression levels of pAKT and mTOR were not down-regulated compared with untreated KYSE30 ESCC cell line.Flow Cytometry:Afatinib treatment on KYSE30 ESCC cell line led to G₀/G₁ phase arrest.Conclusion:Afatinib has more powerful antitumor activities than erlotinib in vitro and in vivo,inhibits the growth of KYSE30 ESCC cell line by down-regulating the expression levels of pEGFR-pERK signaling pathway and might become a potential molecular targeted therapy to ESCC with EGFR amplification or mutantion.