Experimental Study On Influence Of Bufalin In The Metastatic Of Esophageal Squamous Carcinoma Cell Line

Objective: Esophageal cancer is one kind of malignant tumor in theesophageal epithelium. It accounts for2%of all malignant tumors in theworldwide. Some evidences show that there are high rate incidence ofesophageal cancer in our country. Until now, the treatment of esophagealcancer is surgical with or without adjuvant radiotherapy or chemotherapy.Although there are several methods to treat esophageal cancer, the mortalityrate is still high. Most of the patients already have transfer symptoms whenthey go to outpatient. The transfer of esophageal cancer has an importanteffect on survival rate.The occurrence of the tumor is more than one gene involved in long-termcontinuous process, which involves a number of signaling transductionpathways. Extracellular signal-regulated kinase (ERKs) have abnormalexpressed in30%of human tumors,the basic signal transduction isRas→Raf→MEK→ERK. ERKs transducts signal to the nucleus, involves inregulating cell proliferation, differentiation, apoptosis and invasion. If onefactor in this pathway be activated abnormally, the whole pathway willexpress disorder. Raf-1as one of important factor in the ERK pathway, and itcan be activated by Ras and variety of protein factors products. Raf-1kinasephosphorylates and activates MEK (MEK1and MEK2). MEK phosphorylatesand activates ERK. Research has show that Extracellular signal-regulatedkinase (ERKs) play an important role in esophageal cancer.Bufalin is extracted from toxin of Bufo bufo gargarizans Cantor. Therehave been reported that it has inhibitory effects on esophageal cancer.However,the role of anti-metastasis and the underlying mechanism remainelusive. Our experiment performs different concentration Bufalin to cultureesophageal cancer cell strain (TE13). In order to investigate that the effect and mechanism of Bufalin in TE13cell strain.Methods: The experiment based on esophageal carcinoma cell strainTE13culture. Wound Healing assay was perform to found effect of Bufalin oncell migration and cell chemotaxis; chemotaxis of TE13with differentconcentration Bufalin was investigated by Boyden. Western blot andimmunocytochemistry were performed to detect the expression of Raf-1andP-Raf-1in TE13with different concentration Bufalin.Results:1MTT method showed that Bufalin inhibited the growth of esophagealcancer cell lines TE13significantly. Bufalin in the100nM/L and below,under the action of the drug concentration, cell viability greater than50%2The influence of different Bufalin on migration of TE13cell linesWound Healing assay showed that the inhibition of cell migrationincreased with the Bufalin concentration increasing after24hours; Boydencabin showed that the migration of cells through the transwell significantlyreduced with the Bufalin concentration increasing (10nM/L,25nM/L,50nM/L,100nM/L)(P＜0.05); no significant differences between in thecontrol and treatment group(10nM/L)(P＞0.05)。3The influence of different Bufalin on expression of Raf-13.1The expression of Raf-1in TE13cell with different concentrationBufalin was detected by western blot. The result shows that there are nosignificant differences between control group and test group. The expressionof P-Raf-1in TE13cell with different concentration Bufalin was also detectedby western blot. The result shows that there are decreased between controlgroup and test group(25nM/L,50nM/L,100nM/L).3.2The expression of Raf-1in TE13cell with different concentrationBufalin was detected by Immunocytochemistry. The result shows that thereare no significant differences between control group and test group. Theexpression of P-Raf-1in TE13cell with different concentration Bufalin wasalso detected by Immunocytochemistry. The result shows that there aresignificant differences between control group and test group(25nM/L, 50nM/L,100nM/L). This resule are consistent with the above conclusions.Conclusion:1. Bufalin has vital effect on inhibiting the migration of TE13cell strain.The inhibition effect is different with different concentration of Bufalin.2. Bufalin has vital effect on inhibiting the chemotaxis of TE13cell strain.The inhibition effect is different with different concentration of Bufalin.3. Bufalin has no effect on inhibiting the expression of Raf-1, but it haseffect on inhibiting the expression of p-Raf-1in TE13cell strain. Theresult shows that ERK pathway was blocked when Raf-1was inhibited,and then it can inhibit the TE13cell strain metastasis.