| Summary: | 碩士 === 國防醫學院 === 微生物及免疫學研究所 === 98 === Recently, cancer stem cells (CSCs) have been shown to be an important player in tumorigenesis and tumor progression and they may serve as a potential target in cancer therapy. Epithelial-mesenchymal transition (EMT) appears to be actively involved in embryogenesis and development. More recently, it has been demonstrated that EMT may induce transformation of cancer cells that present stem-cell like properties. It has been demonstrated that 3 major transcriptional factors, namely Snail, Twist and Slug, are actively involved in EMT in a variety of human cancers. In view of lack of study on stem cell biology or EMT in esophageal squamous cell carcinoma, we aimed to generate cancer stem cell-like cells from EMT with characterization of CSCs properties in vitro. Hopefully, by establishing this EMT model, we are able to provide a platform for further understanding of tumor biology, to shade some light on the sorting of potential outcome predictors and to develop potential therapeutic targets for ESCC.
A lentivirus system was used to transfect EMT transcription factors such as human Twist and Snail into KYSE 170 and KYSE 510 esophageal squamous cell carcinoma (ESCC) cell lines. Immunoblot was used to determine expression of EMT associated markers (ie Vimentin, E-cadherin, Fibronectin and N-cadherin). Migration and invasion assays were conducted in Snail and Twist-transfected cells with a comparison with their vector controls. Moreover, we used cytotoxicity (MTT) and cell proliferation assay to determine cell growth properties. In addition, sphere formation assay and flow cytometry (FCM) were employed to characterize in part stem cell properties while examining expression of stemness gene (ie Nanog, Oct4, ABCG2 and Sox2) by real-time polymerase chain reaction (PCR).
After transfection of Snail and Twist into KYSE 170 and 510 cell lines, mesenchymal markers Vimentin, Fibronectin, N-cadherin increased, whereas epithelial marker E-cadherin decreased. In addition, migration and invasion ability increased after Snail and Twist-transfection in both of the investigated ESCC cell lines with a decrease in chemosensitivity to cisplatin, taxol, 5’ FU and a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA). Besides, Snail and Twist-transfected ESCC cells presented a significant increase in expression of stemness genes and cell surface marker CD44 with increased sphere formation.
Our results suggested that Snail or Twist transfection induced EMT in selected ESCC cell lines that appeared to harbor cancer stem cell-like properties.
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