The Mechanism Research On EGFR Inhibitor Suppressing Proliferation Of Human Esophageal Cancer Cells

Objective:Esophageal cancer is one of the most common human cancers of digestive tract. The incidence of esophageal cancer is eighth of the morbidity of global carcinoma, and the sixth of the mortality of global carcinoma. Esophageal cancer occurs frequently in China. Rencent years, the therapy of esophageal cancer has mainly involved surgery followed by adjuvant radiotherapy or chemoradiotherapy. According to the foundings in following-up after treatment, the five-year survival rate is not significantly increased. The signal transduction pathway regulates many physiological processes including cancer cell growth、proliferation、metabolism、 transform and invasion, and so on; change the activity of molecular targets of the signal pathway could inhibit the growth of the esophageal cancer. Many molecular targets in the signal pathway have been changed in recent years’ foundings. The scholars focused researches on the EGFR/Ras/Raf/MEK/Erk signal pathway and PI3K/PTEN/Akt/mT0R signal pathway. Gefitinib, a protein tyrosine kinase inhibitor of a selective EGFR, has a substantial effect in the maliganacies which have EGFR mutation, the maliganacies are lung、breast、colon and prostate cancer. But it is not clearly that worked on esophageal cancer. Our study observed the cell toxicity, cell proliferation, the influence of soft-agar colony formation ability, and its influence on the growth of renograft tumor by comparing the esophageal cancer cell line EC9706and KYSE450cells treated with the new EGFR receptor inhibitor, and further discusses the influence of the EGF-Akt and the EGF-ERK1/2signal pathway in KYSE450cell which treated with the new EGFR receptor inhibitor.Materials and methods:1. The drug concentration which about80%cell viability of KYSE450treated with the new EGFR receptor inhibitor has preliminary been selected through the cytotoxicity experiment.2. Cell proliferation assay with different concentrations of the new EGFR receptor inhibitor to test the proliferation of KYSE450cell at0,24,48,72, and96h, and to compare with the results with the cell proliferation treated with gefitinib or erlotinib.3. Soft-agar colony formation assay to observe the colony-forming ability of KYSE450cells treated with different concentrations of the new EGFR receptor inhibitor, and to compare to the results with the cells treated with gefitinib and erlotinib.4. Establishing cancer xenograft model using KYSE450cells, and treated them with different doses of the new EGFR receptor inhibitor and gefitinib through intra peritoneal injection, and observed the anti-tumor effects, and compare with each other.5. Western blotting detected EGF-Erk and EGF-Akt signaling pathway changes of the new EGFR receptor inhibitor on KYSE450cells, and compare with gefitinib and erlotinib.Result:1. The cytotoxicity experiment results showed that25、50、100、250、500nM the new EGFR receptor inhibitor on KYSE450cells at48h, the cell viability was above80%. 2. Cell proliferation results showed that compared with the control group,25、50、100、250、500nM the new EGFR receptor inhibitor on KYSE450cells at24,48,72and96h were able to inhibit the KYSE450cell proliferation (P<0.05) and in a dose-dependent manner.3. Soft-agar colony formation assay results showed that various concentrations of the new EGFR receptor inhibitor on KYSE450cell clones were significantly reduced compared with the control group, the difference were statistically significant (P<0.05).4. The esophageal xenograft model results showed that the difference about tumor size and weight between high-dose the new EGFR receptor inhibitor group and the solvent control group were statistically significant (P<0.05); The tumor growth inhibitory rate of low-dose the new EGFR receptor inhibitor group is36.35%, the high-dose group is82.4%, the high-dose gefitinib group is70.26%. The difference between high-dose the new EGFR receptor inhibitor group and the solvent control group were statistically significant (P<0.05).5. Western blotting found that the new EGFR receptor inhibitor reduced KYSE450cells Erkl/2and Akt phosphorylation level with the increased concentration of the new EGFR receptor inhibitor.Conclusions:1. The new EGFR inhibitor supresses KYSE450cell proliferation both in vivo and in vitro.2. The new EGFR receptor inhibitor supresses KYSE450cells proliferation through reduced its EGF-Erkl/2and EGF-Akt phosphorylation level.