Activating Transcription Factor 5 Mediates A Multidrug Resistance Phenotype Of Esophageal Squamous Cell Carcinoma Cells Through Transactivation Of STAT3 Expression

BackgroundMultidrug resistance is usually one of the main causes for failure of chemotherapy against malignant tumors,including esophageal squamous cell carcinoma.In the past few decades,the efficacy of chemotherapeutic drugs such as cisplatin and paclitaxel in the treatment of esophageal cancer has not been satisfactory.However,the occurrence of primary or secondary MDR has often limited the use of various chemotherapeutic drugs in clinical practice.In recent years,MDR has been found to be associated with gene expression or activation and microRNA in esophageal cancer.However,the complex mechanism of esophageal cancer MDR is far from clear.Several studies on cell stress response suggest that stress resistance can promote tumor cells to survive in adverse environments,Some studies also suggest that stress factors play a role in tumor drug resistance.In recent years,some studies have found that the expression of ATF5 is often up-regulated in cell stress response,which suggests that ATF5 is a stress-related gene.However,there are relatively few studies on ATF5 and tumor at present.The expression of ATF5 in squamous cell carcinoma was significantly higher than that in paracancerous tissue,but the expression and function of ATF5 in esophageal squamous cell carcinoma was not clear.The role of ATF5 in ESCC MDR is also not reported.AimsTo study the role of ATF5 in the pathogenesis of esophageal squamous cell carcinoma(ESCC)and its possible molecular mechanism.Methods1.The role of ATF5 in MDR of esophageal carcinoma1)the expression of ATF5 protein was regulated by transient transfection of plasmid into esophageal carcinoma cells,and ATF5 protein expression groups with different expression levels were constructed.2)the expression of ATF5 protein was detected by Western blot assay.3)the sensitivity of esophageal cancer cells with high expression of ATF5,control group and low expression group to different drugs in vitro was detected by MTT assay.4)DAPI staining was used to detect the difference of apoptosis induced by paclitaxel and cisplatin in cells of different ATF5 protein expression groups.5)using plate clone formation assay to detect the difference of cell proliferation in different ATF5 protein expression groups after the action of Paclitaxel and cisplatin.2.The transcription study of the relationship between STAT3 and ATF51)Eca-109 cells of esophageal carcinoma were transfected with liposome and three groups of cells with different ATF5 expression levels were constructed.2)Western blot was used to detect the expression of ATF5 and STAT3 protein.3)correlation coefficient was used to analyze the correlation between ATF5 expression in different Eca109 cells and STAT3 protein expression in different ATF5 expression levels.3.The role of STAT3 in ATF5 induced MDR in esophageal squamous cell carcinoma1)the expression of STAT3 protein was regulated by the cells transfected to construct the cells with different STAT3 expression levels in the cells with high expression of ATF5 protein.2)the Western Blot test method was used to detect the expression of STAT3 protein in different groups.3)using plate clone formation assay to detect the difference of plate clone formation between control group and siSTAT3 group after down-regulation of STAT3 expression in esophageal carcinoma cells with high expression of ATF5.4)MTT assay was used to detect the drug sensitivity of esophageal cancer cells with high expression of ATF5 after regulating the expression of STAT3 and in combination with Paclitaxel and cisplatin in the high expression group of STAT3,the control group and the low expression group of STAT3.5)DAPI staining and Flow cytometry assay were used to detect the difference of apoptosis in Eca109 esophageal carcinoma cells with high expression of ATF5 after down-regulation of STAT3 and combined with drugs.Results1.ATF5 protein can promote the development of multidrug resistance in esophageal squamous cell carcinoma,and blocking the expression of ATF5 protein can effectively reverse the resistance of esophageal squamous cell carcinoma to chemotherapeutic drugs.1)three groups of Eca109 esophageal carcinoma cells with different ATF5 protein expression levels were successfully constructed.Western blot was used to verify the effect of transfection.2)MTT showed that up-regulation of ATF5 protein expression could increase the resistance of ESCC cells to paclitaxel and cisplatin,while down-regulation of ATF5 protein expression could decrease the resistance of ESCC cells to paclitaxel and cisplatin.3)DAPI staining showed that the number of apoptotic cells of overexpression of ATF5 protein decreased under the action of paclitaxel and cisplatin,but the number of apoptotic cells of low expression of ATF5 protein increased under the action of paclitaxel and cisplatin.4)the results of plate clone formation showed that upregulating the expression of ATF5 protein increased the tolerance of Eca109 cells to paclitaxel and cisplatin,and down-regulating the expression of ATF5 protein decreased the tolerance of Eca109 cells to paclitaxel and cisplatin.2.The stress-related STAT3 expression may transactivated by ATF51)Western blot detected high expression of STAT3 protein in Eca109 cells with high ATF5 expression,and low STAT3 protein expression in Eca109 cells with low ATF5 expression.2)correlation coefficient analysis showed that there was a positive correlation between ATF5 expression in different Eca109 cells and STAT3 protein expression in different ATF5 expression levels.3.STAT3 plays a important role in ATF5 induced MDR in esophageal squamous cell carcinoma1)The results of plate clone formation experiment showed that after down-regulation of STAT3 expression,the number of flat plate clones of Eca-109 or TE-1 esophageal cancer cells under the action of CDDP was significantly reduced compared with that of control group.2)MTT assay showed that the survival rate of STAT3 overexpression group was higher than that of control group,while that of low expression group was lower than that of control group.DAPI assay showed that the number of cells with nuclear staining and fragmentation was significantly higher after treatment with cisplatin for 36 h.DAPI assay showed that ATF5 transfected with siSTAT3 overexpressed Eca109 esophageal carcinoma cells in paclitaxel.After 36 h of cisplatin treatment,DAPI staining showed that the number of cells which transfected with siSTAT3 with nuclear staining and fragmentation was significantly increased after treated with paclitaxel and cisplatin for 36 h.Flow cytometry assay showed apoptosis of esophageal carcinoma cells with high expression of ATF5 increased after siRNA down-regulation of STAT3 and treated with cisplatin.Conclusions1.ATF5 mediates the occurrence of MDR in esophageal squamous carcinoma cells.ATF5 as a target provides a novel approach to reverse MDR in esophageal squamous cell carcinoma.2.STAT3 may be a target gene for transcriptional activation of ATF5.3.STAT3 is involved in and plays a important role in the pathogenesis and development of esophageal squamous cell MDR induced by ATF5.4.STAT3 may also plays a important role in the ATF5-induced esophageal squamous carcinoma MDR and this role might be mediated partly through STAT3 expression.