The Effects Of Blocking Notch Signal Pathway By DAPT On The Proliferation And Apoptosis In U251 Human Glioma Cell Line

Object: To investigate the effects of blocking Notch signal pathway byγ-secretase specific inhibitor DAPT on the proliferation and apoptosis in U251 human glioma cell line harboring constitutive active Notch and the underlying mechanisms.Methods:The glioma cell line U251 was treated with DAPT in different doses. Then the morphologic changes of the cells were observed by the inverted microscope.The cell proliferation were detected by MTT assay. Cell cycle was detected by flow cytometry. U251 glioma cells were inoculated into endermic tissues of BALB/c mouse, and the xenograft tumor growth was observed. The changes of cell morphology were observed by transmission electron microscope (TEM). Apoptotic cells were detected by DNA gel electrophoresis and TUNEL labeling methods. The expression of Notch1,Hes1 and Hes5 protein was studied by immunocytochemistry.Results:Compared with the control group, after the cells were incubated with DAPT, obvious morphologic changes of U251 cells were observed; The growth and proliferation of human glioma cells decreased significantly after DAPT treatment (P <0.01), DAPT produced concentration dependent antiproliferative effects on U251 cells. DAPT could block cell cycle at G0 / G1 phase. In the nude mice model , the growth of xenograft tumors was suppressed obviously(P <0.01). Under transmission electron microscope, we saw that the cells of U251 treated by DAPT, becomes small and shrinkage with the separate chromatin and the concentrated cytoplasm and the nuclear is splited into the part. Typical DNA ladder were seen from gel electrophoresis , Apoptosis occured in the late stage was identified by TUNEL labeling methods respectively. Immunocytochemistry showed that protein expression of Notch1,Hes1 and Hes5 in U251 human glioma cells decreased significantly after DAPT treatment(P <0.01). Conclusions:1. DAPT could significantly inhibit the proliferation of U251 cells in dose dependent manner in vitro.The inhibition mechanisms were associated with blocking cell cycle at G0/G1 phase. 2.DAPT could significantly result in reduction in U251 cells line tumor exongraft volume in vivo. 3. DAPT could induce the apoptosis of U251 cells in vitro. 4. DAPT could efficiently inhibit growth and induce apoptosis in U251 cells, which may be related with the down-regulation of Notch1,Hes1 and Hes5 protein expression.