| Objective: It is believed that the Notch signaling pathway may play apivotal role in cellular differentiation, proliferation and apoptosis. However,the function of Notch1signaling pathway in gastric cancer stem cells islargely unknown. This study aimed to delineate the role of the Notch1signaling pathway in gastric cancer stem cells and Epithelial-MesenchymalTransition (EMT).Methods: The expression levels of Notch1in gastric cancer cells(MKN45and AGS), normal gastric mucosal cells (GES1)，45cases ofgastric carcinoma tissue and25cases of normal gastric tissue were analysedby RT-PCR and Western blot. We used flow cytometry to isolate CD44+andCD44-cells from the human gastric cancer cells line MKN45, then detectedstemness properties by in vivo tumorigenicity assay and in vitro spheroidcolony formation assay, sensitivity to the chemotherapy drug5-Fu was testby MTT, expression levels of Notch1and Hes1were measured by Westernblot. To further explore the role of the Notch1signaling pathway in gastriccancer stem cells, CD44+and CD44-MKN45cells were treated byγ-secretase inhibitor DAPT, and then we detected expression levels of Hes1and EMT, cancer stemness properties and capability of proliferation, metastasis and invasion.Results: Using RT-PCR and Western blot analysis, we found that theexpression of Notch1was increased in gastric cancer cell lines compared tothe normal gastric mucosa cell line. We also found that Notch1expressionwas down-regulated in the non-metastatic-derived gastric cancer cell linecompared to the metastatic-derived cell line. Moreover, Notch1expressionswere significantly increased in gastric cancer tissues compared to humanadjacent normal mucosa tissues, and Notch1expressions in gastric cancerpatients with metastases were significantly higher than those in gastriccancer patients without metastases. Then we found that CD44+MKN45cells displayed the characteristics of cancer stem cells (CSCs) and exhibitedhigher Notch1expression than CD44-MKN45cells. DAPT treatmentinhibited the expression of the Notch1downstream target Hes1and EMTmarkers, impaired the properties of CSCs and suppressed the invasion andproliferation capabilities of CD44+MKN45cells, but those changesdescribed above were not found in CD44-MKN45cells. Moreover,intraperitoneal treatment with DAPT effectively inhibited the growth ofCD44+MKN45cells xenograft tumors.Conclusion: Our data suggested that CD44+MKN45gastric cancercells possessed the characteristics of CSCs and that the Notch1signalingpathway played a critical role in the maintenance of CSCs and EMT.Moreover，treatment of γ-secretase inhibitor DAPT could block Notch1 signaling pathway, which impaired gastric cancer stemness properties andcapability of proliferation, metastasis and invasion. |
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