Inhibitory Effect Of Sorafenib On Growth Of Human Esophageal Cancer Cells EC9706

Background and Purpose:Esophageal cancer is one of the most common gastrointestinal tumor, the annual global incidence of about 482000 new cases and about 407000 cases of death for a year; the incidence of Chinese thick and crude esophageal cancer mortality rate ranked first in the world; in Chinese malignant tumor, the incidence of esophageal cancer rate ranks fourth, with mortality ranking the fifth. Esophageal cancer in China has been dominated by squamous cell carcinoma of the esophagus, and esophageal adenocarcinoma incidence rate had less proportion. The treatment mode of esophageal cancer is decided by the stage, staging in early or middle part of esophageal cancer patients can choose operation or radiotherapy combined with chemotherapy, but advanced patients can only choose chemotherapy. Chemotherapy is one of the important means in advanced esophageal carcinoma treatment, but the total effect is not satisfying.Sorafenib as a small molecule multikinase inhibitor, can effectively inhibit the extracellular signal regulated kinase and Raft mitogen activated protein kinase(MEK)/extracellular signal regulated kinase(ERK) signal transduction pathway. At present, sorafenib has been used in patients with advanced renal cell carcinoma and advanced hepatocellular carcinoma. A large number of phase I, II clinical trial found that sorafenib had therapeutic effect on malignant tumor like lung cancer and malignant melanoma. At present, the effect of sorafenib on esophageal cancer has not been reported, and this study was designed to investigate the inhibitory effect and mechanism of sorafenib on human esophageal squamous cell carcinoma cell EC9706.Methods1.Cell scratch test: In the sorafenib final concentration of 3μmol/L and 6μmol/L dosing group, control group without dosing, after 24 h drugs effect acting cell morphology change was observed on an inverted microscope sampling, taking photos, measuring relative distance of cell migration to the scratch area.2.Hoechst/PI staining: The experimental group according to the sorafenib final concentration of 3μmol/L, 6μmol/L dosing group, control group without medicament. After the cells were cultured for 48 h, observe the change of cellular morphology, sampling, taking pictures on an upright fluorescence microscope.3.The expression level of 5 kinds of gene m RNA were detected by q RT-PCR: The experimental group according to the sorafenib final concentration of 6μmol/L dosing group, control group without medicament. After cells were cultured in 48 h, cells were collected and the m RNA expression of MMP2, MMP9, TIMP1, AKT and Bcl-2 m RNA were detected by q RT-PCR.4.The expressions of 5 kinds of protein were detected by Western blot: The experimental group according to the sorafenib final concentration of 6μmol/L dosing group, control group without medicament. After cells were cultured in 48 h, cells were collected and the expression of AKT, p-AKT, Bcl-2, MMP2 and TIMP1 were detected by Western blot.5.Statistical analysis: We use SPSS 17 software for statistical analysis. Data expressed by X + s, two samples were collected by t test, multigroup were compared with single factor analysis of variance after variance homogeneity test,comparison between groups with LSD-t method, P < 0.05 for the difference had statistical significance.All experiments were repeated at least 3 times. Results1.By cell scratch test, after cultured for 24 h, the relative distance of cell migration into the scratch area in the control group is significantly reduced compared with 3μmol/L group and 6μmol/L group, with significant difference(P < 0.05).2.After the effect of sorafenib EC9706 cells by 48 h, compared with control group, the experimental group(3μmol/L group and 6μmol/L group) red staining cells increase, and with the increase of drug concentration, the apoptosis cells were significantly increased(P < 0.05).3.The effect of the expression level of sorafenib on EC9706 cells of 5 gene m RNA results show that, compared with the control group, drug group(6μmol/L group) can downregulate AKT, Bcl2, MMP2, MMP9 expression, upregulate TIMP1 expression.4.The effects of sorafenib on the expression of 5 protein levels in EC9706 cell :compared with the control group, sorafenib in 6μmol/L group, can significantly decrease the expression of AKT, p-AKT, MMP2, Bcl-2 protein, improve the expression level of TIMP1 protein. ConclusionsSorafenib can upregulate the expression of TIMP1, downregulate the expression of MMP9, AKT, MMP2 and Bcl-2, and inhibit the migration and promote apoptosis/necrosis of esophageal cancer cells EC9706. These may be its anticancer mechanism.