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C-Myc Affects Esophageal Squamous Cancer Sensitivity to Paclitaxel by Regulating BubR1 Expression |
HU Min, SONG Pei-pei, ZHAN Xiao-qin, LÜ Zi-lan, CHEN Chu, SHI Qiong, WENG Ya-guang |
The Key Laboratory of Laboratory Medical Diagnostics in Ministry of Education, Chongqing Medical University, Chongqing 400016, China |
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Abstract 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.
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Received: 15 October 2012
Published: 25 April 2013
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