The Effects Of Bufalin On The Active State Of FAK And Epithelial Mesenchymal Transition In Esophageal Squamous Cell Carcinoma

Esophageal carcinoma is one of the most common malignant tumors in China. One reason of high mortality rate is early metastasis and invasion. Focal adhesion kinase(FAK) is a 125 k D non receptor tyrosine kinase. Studies showed that FAK plays an important role in the development of esophageal carcinoma. In recent years, the role of FAK in tumor stem cells, tumor microenvironment and epithelial mesenchymal transition(EMT) has gradually become the research hotspot. Epithelial mesenchymal transition(EMT) refers to the process of transformation from epithelial cells to mesenchymal cells after being stimulated, while reducing and around the cell matrix adhesion, cytoskeletal remodeling, increased cell motility.Bufalin was extracted from traditional Chinese medicine Venenum Bufonis in a class of digitalis with anti tumor effect. But the specific mechanism of Bufalin in esophageal carcinoma is still not clear.Our laboratory has made a lot of research on the role of Bufalin in Ras/Raf/MEK/ERK signal pathways in esophageal cancer. We found tha Bufalin play the role of anti-tumor through the down-regulation of the active state of ERK. In order to clear the specific mechanism of esophageal cancer occurrence and development better, this experiment through in vivo and in vitro studies, to investigate the effect of Bufalin on esophageal carcinoma in the activation of FAK and epithelial mesenchymal transition, in order to further explore the mechanism of Bufalin in human esophageal cancer tumor.Objective: To investigate the effects of Bufalin on the active state of FAK and epithelial mesenchymal transition in esophageal squamous cell carcinoma.Methods: 1 The m RNA expression of FAK, E-Cadherin, Vim in different concentrations of Bufalin in ECA109 cells were examined by RT-PCR.2 The protein expression of FAK, E-Cadherin, Vim and the active state of FAK in different concentrations of Bufalin in ECA109 cells were examined by Western Blot method.3 The scratch healing experiment and Transwell chamber experiment were used to detect migration and invasion ability in different concentrations of Bufalin in ECA109 cells.4 36 nude mice transplanted tumor model with human esophageal squamous cell carcinoma cell line ECA109 were randomly divided into 6 groups, namely model group, Bufalin low dose group(0.5mg/kg, BL), middle dose of Bufalin group(1mg/kg, BM), Bufalin high dose group(1.5mg/kg, BH), PF562271 group(FAK inhibitor), and PF562271 combined with high dose of Bufalin group(BP), intraperitoneal injection of drug 30 days continuously. Nude mice diet, mental status, weight, and tumor size data were observed and recorded during the treatment.5 After the medication, all the mice were sacrificed, measuring the size of tumors and calculating the tumor inhibition rate.6 The characteristics of HE staining in orthotopic transplanted tumor tissues of nude mice.7 The m RNA expression of FAK, E-Cadherin, Vim in nude mice orthotopic transplanted tumors were examined by Real time-PCR.8 The protein expression of FAK, E-Cadherin, Vim and the active state of FAK in nude mice orthotopic transplanted tumors were examined by immunhistochemical method.Results: 1 Effect of Bufalin on the expression and active state of FAK in esophageal squamous cell carcinoma ECA109 cellsRT-PCR results showed that Bufalin decreased the m RNA expression of FAK. The m RNA expression of FAK were 1.276±0.046, 1.154±0.027, 0.619±0.026, 0.556±0.0172, 0.439±0.022, 0.221±0.0206, 0.210±0.0285 in the positive control group, 20, 40, 60, 80, 100 nmol/L Bufalin and PF562271 group respectively. The m RNA expression of FAK were decreased in medicine intervention groups compared with the positive control group, the differences were statistically significant(P<0.05). Western Blot results showed that Bufalin decreased the active state of FAK. The protein expression of FAK were 1.255±0.111, 1.357±0.050, 1.177±0.065, 1.278±0.099, 1.239±0.196, 1.199±0.106, 1.188±0.094 in the positive control group, 20, 40, 60, 80, 100 nmol/L Bufalin and PF562271 group respectively, the differences were not statistically significant(P>0.05). The active state of FAK were 1.315±0.020, 1.159±0.058, 1.002±0.069, 0.773±0.027, 0.589±0.027, 0.410±0.026, 0.379±0.044. The active state of FAK were decreased in medicine intervention groups compared with the positive control group, the differences were statistically significant(P<0.05).2 Effect of Bufalin on epithelial mesenchymal transition process in human esophageal squamous cell carcinoma ECA109 cellsRT-PCR results showed that Bufalin increased the m RNA expression of E-Cadherin, decreased the m RNA expression of Vim. The m RNA expression of E-Cadherin were 0.231±0.016, 0.435±0.048, 0.568±0.034, 0.669±0.025, 0.842±0.031, 0.939±0.036, 0.948±0.027 in the positive control group, 20, 40, 60, 80, 100 nmol/L Bufalin and PF562271 group respectively. The expression of E-Cadherin were increased in medicine intervention groups compared with the positive control group, the differences were statistically significant(P<0.05). The m RNA expression of Vim were 0.852±0.051, 0.682±0.025, 0.567±0.034, 0.431±0.047, 0.300±0.016, 0.212±0.024, 0.190±0.022. The expression of Vim were decreased in medicine intervention groups compared with the positive control group, the differences were statistically significant(P<0.05). Western Blot results showed that Bufalin increased the protein expression of E-Cadherin, decreased the protein expression of Vim. The protein expression of E-Cadherin were 0.351±0.009, 0.459±0.006, 0.515±0.006, 0.619±0.006, 0.756±0.005, 0.859±0.007, 0.867±0.005 in the positive control group, 20, 40, 60, 80, 100 nmol/L Bufalin and PF562271 group respectively. The protein expression of E-Cadherin were increased in medicine intervention groups compared with the positive control group, the differences were statistically significant(P<0.05). The protein expression of Vim were 1.257±0.043, 1.150±0.038, 0.854±0.032, 0.754±0.025, 0.621±0.028, 0.552±0.034, 0.539±0.040, the protein expression of Vim were decreased in medicine intervention groups compared with the positive control group, the differences were statistically significant(P<0.05).3 Effect of Bufalin on cell migration and invasion abilityThe results indicated that Bufalin inhibited cell migration and invasion in ECA109 cells. Compared with the positive control group, 20, 40, 60, 80, 100 nmol/L Bufalin and PF562271 group scratch test results showed that the scratch distance were increased from 1.156±0.118 to 1.556±0.249, 2.981±0.167, 3.685±0.121, 4.614±0.170, 5.316±0.104, 5.795±0.178, cell numbers of Transwell migration experiment crossed the basement membrane decreased from 107.00±8.19 to 78.67±3.06, 61.67±3.06, 42.67±3.512, 24.33±2.517, 10.33±3.215, 9.00±2.65, the cell numbers of Transwell invasion experiment decreased from 127.67±8.02 drop to 102.33±4.51, 87.33±7.10, 73.00±4.58, 57.33±2.52, 39.00±3.61, 37.33±2.52, the differences were statistically significant(P<0.05).4 Each group of transplantation tumors in nude mice during the experimental process(Model, BL, BM, BH, PF562271, BP group) have no difference in diet and mental state, there were not statistically significant difference in body weight before and after treatment(P>0.05).5 Effect of Bufalin on growth of transplantation tumor in nude miceTumor volume of Each group(Model, BL, BM, BH, PF562271, BP group) were 2.430±0.052cm3, 2.378±0.042cm3, 1.805±0.052cm3, 1.722±0.072 cm3, 1.703±0.062cm3, 1.128±0.064cm3. The tumor volume decreased gradually, compared with the model group, the difference of BM, BH, PF562271, BP group were statistically significant(P<0.05). The medicine intervention groups(BL, BM, BH, PF562271, BP group) of tumor inhibition rates were 4.4%, 45.6%, 63.5%, 65.2%, 71.1%.6 The characteristics of HE staining in orthotopic transplanted tumor tissues of nude miceMorphological observation results in nude mouse orthotopic transplanted tumor in each groups: HE staining sections were observed through microscope. The tumor tissues of nude mice orthotopic transplanted tumor were poorly differentiated squamous-cell carcinoma(PDSCC), the heteromorphism was obvious in cancer cells, karyomegaly and anachromasis, cell morphological were various, arranged in solid nests of unequal size, the nuclear chromatins showed coarse granular, nucleolus were obvious, infrequent ofintercellular bridge and keratosis.7 Effect of Bufalin on FAK, the active state of FAK and EMT of transplantation tumor in nude miceReal time-PCR results showeded that Bufalin decreased the expression of FAK, increased the expression of E-Cadherin, decreased the expression of Vim. The m RNA expression of FAK in each group(BL, BM, BH, PF562271, BP) were 0.956±0.015, 0.677±0.041, 0.516±0.019, 0.480±0.015, 0.343±0.022, the expression of FAK were decreased. The m RNA expression of E-Cadherin were 1.137±0.140, 1.840±0.095, 2.593±30.127, 2.600±0.151, 3.245±0.100, the expression of E-Cadherin were increased. The m RNA expression of Vim were 1.004±0.033, 0.791±0.027, 0.540±0.026, 0.501±0.015, 0.399±0.023, the expression of Vim were decreased. Compared with the Model group, the difference of BM, BH, PF562271, BP group were statistically significant(P<0.05).Immunohistochemical results showeded that Bufalin decreased the expression and activation of FAK, increased the expression of E-Cadherin, decreased the expression of Vim. The expression of FAK in each groups(Model,BL, BM, BH, PF562271, BP) were 49.20, 46.70, 36.35, 24.60, 20.10, 6.05, the expression of FAK were decreased; the active state of FAK were 46.90, 40.50, 31.15, 26.00, 22.80, 15.65, the activation of FAK were decreased; the protein expression of E-Cadherin in each groups were 8.75, 14.00, 24.40, 40.60, 42.5, 52.75, the expression of E-Cadherin were increased; the protein expression of Vim in each groups were 51.00, 47.90, 36.40, 21.10, 20.20, 6.40, the expression of Vim were decreased, compared with the Model group, the difference of BM, BH, PF562271, BP group were statistically significant(P<0.05).Conclusions:1 Bufalin can inhibit the migration and invasion of ECA109 cells.2 Bufalin can inhibit the growth of esophageal squamous cell carcinoma in nude mice transplanted tumor.3 Bufalin can inhibit the activation of FAK and epithelial mesenchymal transition process in esophageal squamous cell carcinoma, indicated that Bufalin may inhibit the epithelial mesenchymal transition process through suppressing FAK.