C-Myc Affects Esophageal Squamous Cancer Sensitivity to Paclitaxel by Regulating BubR1 Expression

To explore the relationship between C-Myc and mitotic checkpoint protein BubR1 expression and possible influence of C-Myc on paclitaxel drug effects.Immunohistochemistry assay was explored to detect C-Myc and BubR1 expression in twenty-three clinical esophageal squamous cancer samples.And Western blot was applied to detect and compare C-Myc and BubR1 expression levels in three esophageal squamous cancer cell lines,ECA-109,KYSE150 and KYSE180.The correlation between C-Myc and BubR1 protein expression was analyzed.About 2000bp fragment upstream human BUB1b gene was cloned and inserted into pSEAP2 promoter reporter vector to construct pSEAP2-BubR1-P2000.Then pSEAP2-BubR1-P2000 was transfected into three cell lines for promoter activities detection(ALP activity).ALP activity was detected in ECA-109 cells both overexpressing C-Myc and transfected pSEAP2-BubR1-P2000.Western blot assay was used to detect the effect of C-Myc inhibitor 10058-F4 on BubR1 expression.MTT assay was applied to detect cell viability under gradient paclitaxel exposure after C-Myc suppression.DAPI staining was explored for apoptotic cell count in C-Myc inhibitor group,low concentration paclitaxel(100nM) group and combination of C-Myc inhibitor and paclitaxel,respectively.The results showed that C-Myc expression positively correlated with BubR1 expression both in clinical esophageal squamous cancer tissues and in esophageal squamous cancer cell lines.Cells with high C-Myc expression showed higher BubR1 promoter activity and overexpression of C-Myc in ECA-109 cells can further enhance BubR1 promoter activity.Specific C-Myc inhibitor 10058-F4 can effectively down-regulate BubR1 expression and decrease cell viability under gradient paclitaxel exposure.DAPI staining result exhibited that combination of 10058-F4 and paclitaxel induced significantly more mitotic cells than single usage of 10058-F4 or paclitaxel.In esophageal squamous cancer cells,C-Myc can regulate mitotic checkpoint protein BubR1 expression and related with paclitaxel sensitivity.C-Myc may reduce esophageal squamous cancer response to paclitaxel by up-regulating BubR1 expression.