Effects Of DAPT On The Notch4、DLL1 And ALDH1 Of Colorectal Cancer Cell Line HT-29

Objective:In this study,we investigated that the Notch signal pathway blockers gamma secretase inhibitors(GSIs) DAPT acts on the colorectal cancer cell line HT-29,Which preliminarily explores the expression and correlation of colorectal tumor stem cell marker ALDH1 and Notch signaling pathways Notch4 receptor and ligand DLL1 in this cell,so as to provide theoretical evidence for the treatment of colorectal cancer. Methods:The object of study was the HT-29 colorectal cancer cell lines.First we cultivated the HT-29 cells and extracted phase of cell growth index for the followin g step test respectively in the conditions for 37℃、5%CO2、20%O2 and 95% humi dity incubator.(1)Tested the expression of ALDH1,Notch4,DLL1 protein and the expression level o f ALDH1,Notch4, DLL1 protein of the different concentrations of DAPT to the HT-29 by cell immunohistochemistry,and quantified measured by image analysis system s.(2)The influence of different concentrations of DAPT(0, 5, 10, 20, 40, 80μmol/L)on the proliferation of HT-29 was measured by MTT and observed HT-29 cell morph ology by inverted microscope.(3)The m RNA and protein expressions of ALDH1,Notch4 and DLL1 in DAPT actio n HT-29 cells were analyzed by real-time PCR and western blot. Results:(1)The results showed that the ALDH1,Notch4,DLL1 in HT-29 cells were expressed by cell immunohistochemical.The more and longer use of DAPT on HT-29, the higher restrain rate on the proliferation of HT-29 was observed(P<0.05).(2)After stimulation of HT-29 by 0,5,10,20, 40, 80μmol/L DAPT,the HT-29 cells presented typical morphological features of apoptosis by microscope.The proliferation inhibiting of DAPT were 0%、0.22%、5.85%、11.41%、13.82%、18.59%for 24 h by MTT;it was 0%、6.06%、16.8%、25.44%、44.25%、56.73% for 48h;it was 0%, 6.9%,4.86%, 27.6%,4.86% and 67.78% for72 h.With the increase of DAPT concentration and time,the proliferation inhibition rate of HT-29 cell increased significantly,the experimental group compared with control group was statistically difference(P<0.05).The 40 and 80umol/L DAPT in 48h、72h were compared with 24 h,the difference was statistically significant(P<0.05).(3)The mRNA and protein expression and relevance of ALDH1, Notch4 and DLL1,The 20 and 40μmol/L DAPT acted on HT-29 for 48 hours:The m RNA and protein of ALDH1, DLL1 and Notch4 in HT-29 cells were lower(P<0.05),and has was compared also statistical significance between 20 and 40μmol/L(P<0.05). Moreover, there was pertinence between m RNA and protein expression of ALDH1 and Notch4, and between expression of Notch4 associated with DLL1(P<0.05).But there was no pertinence between m RNA and protein expression of ALDH1 and DLL1(P<0.05).Therefore,The m RNA and protein expression difference statistical significance of ALDH1, Notch4 and DLL1 were same, there may be a mutual relationship between the three factors. Conclusion:(1)Notch4, DLL1 and ALDH1 havewere expressed in HT-29 colorectalcancer cell lines.(2)DAPT could inhibit the proliferation of colon cancer cell HT-29 in vitro, and this inhibit ion may have time and concentration dependence.(3)ALDH1 has wasexpressed highly in the colon cancer cell line HT-29,but when Notch si gnal pathway inhibitor play dapt role, ALDH1 expression with the Notch signaling pathwa y in the NOTCH4 and Dll1 expression downregulation occurs the corresponding reduced.(4)Notch4, DLL1 and ALDH1 may exist in the regulation of Notch signaling pathway in c olon cancer HT-29 cells.