| Objective To isolate, identify and localize the pancreatic cancer stem-like cells using Notch2as surface marker in cell lines or tissues, and to search for methods of targeted therapy on these cells.Methods In this paper, we induced the spheres formation in Panc-1, Bxpc-3, Aspc-1and Cfpac-1pancreatic cancer cell lines by culturing the cells separately in the serum-free conditions supplemented with EGF, IGF-1and FGF-10. Their expressions of self-renewal regulators, OCT4and Nanog, were measured by immunofluorescent staining among five groups:4sorts of pancreatic cancer cell lines and their relevant spheres,15cases of pancreatic cancer tissues and13cases of normal pancreas, and Panc-1cell line after treated by gemcitabine. The Notch2+cells were separated from Bxpc-3and Panc-1cell lines by magnetic activated cell sorting (MACS). Then series of tests would be carried out included that tumorigenicity, which was evaluated via transplantation of these cells into NOD-SCID mice, tumorsphere formation ability in serum-free conditions, expression of sternness related markers, OCT4, Nanog and PDX1, which were measured by immunofluorescent staining, and K-ras mutation detected by DNA sequencing technology. The drug resistance of Notch2+Panc-1cells was estimated after treated with gemcitabine.4cases of normal pancreas and4cases of pancreatic cancer tissues were chosen for immunohistochemistry staining of Notch2. AQP1and AQP5, which are water channel proteins, associated with Notch2were measured by immunofluorescence staining in Bxpc-3cells. Notch signal pathway inhibitors Compound E (CE), Ras signal pathway inhibitors B10, SB239063and U0126were acted on Notch2+Bxpc-3cells respectively. After that, the cell vitalities were measured by CCK-8method. Expressions of OCT4, Nanog, PDX1and AQP5in Notch2+Bxpc-3cells, before and after treatment with CE, were tested by immunofluorescence staining.Results After5to10days culture in serum-free DF12, the float-growing spheres were developed in all the cell lines tested. The expression of OCT4and Nanog in the sphere-forming cells was much higher than their relevant counterparts in cell lines. Furthermore, a high expression was also observed in Panc-1cells after treated by gemcitabine. These sternness related markers were also found in the pancreatic cancer tissues and at much lower level in normal pancreas. Besides, the stem-like spheres in Panc-1were observed to express SAMA6A, a surface marker known to be the OCT4downstream target. The Notch2+pancreatic cancer cells possessed a markedly tumorigenic ability. The positive expressions of core transcription factors (OCT4, Nanog and PDX1) of embryonic stem cells were observed in these Notch2+cells. The Notch2+cells were able to form spheres when cultured in serum-free medium. K-ras mutations were found in these cells. The percentage of Notch2+cells in Panc-1cell line rose after treated by gemcitabine. The Notch2+cells located at centroacinar cells and terminal ductal cells, which expressed AQP1and AQP5, in normal pancreas and pancreatic cancer tissues. The enzyme inhibitor CE, BIO, SB239063and U0126could depress Notch2+Bxpc-3cells in some degree. CE, one of the γ-secretary enzyme inhibitors, could depress the proliferation of Notch2+cells, but had no differentiation-inducing effect on them.Conclusion OCT4and Nanog, two pivotal transcription factors of embryonic stem cells, whose expression in pancreatic cancer was related with cancer stem cells, could be used as markers for self-renewal of pancreatic cancer stem cells. The SEMA6A protein regulated by OCT4may represent an invaluable surface marker for pancreatic cancer stem cells, which worth further research. The Notch2+cells in human pancreatic cancer Bxpc-3and Panc-1cells exhibitd the properties of cancer stem cells. The results supported the hypothesis that pancreatic ductal adenocarcinoma originated from centroacinar cells. The targeted therapy by blocking the Notch and Ras signal pathway shew effect on the putative pancreatic cancer stem cells. |
PDF Full Text Request