Effects of DAPT on proliferation of colon cancer cell line HT-29, expression of ALDH1, Notch signaling pathway receptor and ligand
ZHAO Xiaofang
WANG Hong
Abstract：Objective To observed the effects of γ-secretase inhibitors (GSIs) DAPT on proliferation of colon cancer cell line HT29, colorectal tumor stem cell marker aldehyde dehydrogenase 1 (ALDH1), Notch signaling pathway receptor Notch4 and Notch signal pathway ligand DLL1 expression. Methods HT-29 cells were cultured in vitro, and the cells in the logarithmic phase were used in the experiment. DAPT (0, 5, 10, 20, 40, 80 umol/L) was used to treat HT-29 cells for 24, 48 and 72 h, we observed the cell morphology under inverted microscope, detected the cell proliferation ability by MTT colorimetry (OD value). DAPT (0, 20, 40 μmol/L) was used to treat HT-29 cells for 24 h, the ALDH1, Notch4, DLL1 mRNA was detected by RT-PCR, the ALDH1, Notch4, DLL1 protein was detected using Western blotting and immunohistochemistry. The correlation of ALDH1, Notch4, DLL1 mRNA and protein expression in HT-29 cells was analyzed. Results The proliferation inhibition of DAPT on HT-29 cells was in a dose- and time-dependent manner. The proliferation ability of cells at 48, 72 h was lower than that at 24 h in the 40, 80 umol/L groups (P<0.05). Cells showed typical apoptosis state change after DAPT treatment. The mRNA expression of ALDH1, Notch4 and DLL1 in HT-29 cells treated with 20, 40 umol/L DAPT was lower than that treated with 0 umol/L DAPT (all P<0.05). The protein expression of ALDH1, Notch4 and DLL1 in HT-29 cells treated with 0, 20, 40 μmol/L DAPT was decreased successively (all P<0.05). Immunohistochemical image analysis, the OD value of protein expression of ALDH1, Notch4, and DLL1 in HT-29 cells treated with 0, 20, 40 umol/L DAPT was reduced in turn (all P<0.05). The mRNA expression of ALDH1 and DLL1 in HT-29 cells was positively correlated with Notch4 mRNA expression (r=0.997, 0.998; all P<0.05). The protein expression of ALDH1and DLL1 was positively correlated with Notch4 protein expression in the HT-29 cells (r=0.998, 0.999, all P<0.05). Conclusions After DAPT treatment, the cell proliferation is inhibited and the mRNA and protein expression of ALDH1, Notch4 and DLL1 is down-regulated. The proliferation inhibition of DAPT on HT-29 cells may be related to reducing the cancer stem cell properties and down-regulating Notch signaling pathwayreceptor and ligand expression.
Keywords：colorectal carcinomaHT-29 cellsγ-secretase inhibitors(2S)-NN-(35-fluorophenylacetyl) -L-alanyl-2-phenylglycine tert-Aldehyde dehydrogenase 1tumor stem cellsaldehyde dehydrogenase 1Notch signaling pathwayNotch4DLL1
Publication Date：2017-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 5-8 )