Radiosensitization And Its Mechanism Of Nimotuzumab On Esophageal Cancer Cells With Different EGFR Phenotypes

Background:Esophageal cancer is a common malignant tumor of digestive system,China is a high incidence area of esophageal cancer,accounting for 46.6%of the total number of esophageal cancer in the world,and the incidence and mortality of malignant tumors were the sixth and the fourth.Only early esophageal cancer has a chance of a cure by surgery,but most of them have no typical symptoms and cannot be diagnosed early.For patients with locally advanced esophageal cancer who lost the chance of surgery,radiation therapy has become the primary means.However,the tolerance of esophageal cancer patients is poor,and the local control rate of radiotherapy is limited.Molecular targeted therapy brings new hope for cancer therapy.Epidermal growth factor receptor(EGFR)is one of the most important targets in cancer research.The abnormal activation of EGFR pathway can promote the transformation of normal cells and the production of malignant tumors.The positive rate of EGFR in esophageal squamous cell carcinoma was 40?80%.Nimotuzumab is the first humanized monoclonal antibody targetingEGFR in China.Specific binding to the extracellular domain of EGFR III,blocking the combination of EGFR and transduction of ligands and their downstream signaling pathways,thereby enhancing radiosensitivity of tumor cells,improve the efficacy of radiotherapy and inhibition of tumor growth,metastasis and local recurrence.Currently,Nimotuzumab is currently used in the treatment of head and neck malignant tumors and the effect of Nimotuzumab on the radiosensitivity of esophageal cancer cells is not clear.In this study,we will investigate the radiosensitivity of esophageal cancer cells treated with Nimotuzumab and try to explore the mechanism of radiosensitization.MethodsMTT assay was used to detect the effect of Nimotuzumab on the proliferation of each cell line.We used a clonogenic assay to detect radiosensitization in response to Nimotuzumab.KYSE410,KYSE30 and KYSE520 esophageal cells were treated with three concentrations of 50ug/ml,100 ug/ml and 200ug/ml,for up to 24 hours and then given 0,1,2,4,6,8Gy dose irradiation.The results showed that the concentration of 200ug/ml increased the radiosensitivity of KYSE30 cells compared with control groupnot not treated with Nimotuzumab,and however,the effect of low concentration of monoclonal antibody on the radiosensitivity of KYSE30 cells was not obvious.Nimotuzumab had no significant effect on the radiosensitivity of KYSE410 and KYSE520 cells.Then,We investigated the mechanism of Nimotuzumab on the radiosensitization of esophageal cancer cells.We used Western blot to detect the expression and phosphorylation of EGFR of the three esophageal carcinoma cells KYSE410,KYSE30 and KYSE520.We further examined the key proteins AKT on the PI3K/AKT signaling pathway and the key proteins MAPK on the Ras/Rad/MEK/MAPK signaling pathwayResultsMTT assay showed that the growth inhibition of KYSE410,KYSE30 and KYSE520 cells was not significant after treatment with different concentrations of Nimotuzumab.The results of clonogenic assay showed that the concentration of 200ug/ml increased the radiosensitivity of KYSE30 cells compared with control group not treated with Nimotuzumab,and however,the effect of low concentration of monoclonal antibody on the radiosensitivity of KYSE30 cells was not obvious.Nimotuzumab had no significant effect on the radiosensitivity of KYSE410 and KYSE520 cells.It was found that the expression of EGFR in esophageal carcinoma cell line KYSE30 was high,the degree of phosphorylation was high,the expression of KYSE410 EGFR was high,the degree of phosphorylation was lower,and the expression of KYSE520 EGFR was low.We find the phosphorylation of AKT protein in KYSE30 cells was inhibited by the treatment with Nimotuzumab,and The expression and phosphorylation of AKT protein in KYSE410 cells and KYSE520 cells were not significantly changed.The expression of MAPK protein in the Ras/Rad/MEK/MAPK signaling pathway of the three esophageal carcinoma cells was not significantly changed before and after treatment with Nimotuzumab.Conclusions:Nimotuzumab can increase the radiosensitivity of esophageal cancer cells with high expression of EGFR and high degree of phosphorylation,dose effect relationship exists in sensitization,and the sensitization mechanism may be related to the inhibition of EGFR downstream signal transduction pathway PI3K/AKT.