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.%目的 观察γ-分泌酶抑制剂(GSIs)DAPT对结肠癌细胞株HT-29增殖及结直肠癌肿瘤干细胞标志物乙醛脱氢酶1(ALDH1)、Notch信号通路受体Notch4、Notch信号通路配体DLL1表达的影响.方法 体外培养HT-29细胞,取对数生长期细胞用于实验.分别以0、5、10、20、40、80 μmol/L的DAPT作用于HT-29细胞24、48、72 h后,倒置显微镜下观察细胞形态,用MTT比色法检测细胞增殖能力(以OD值表示).分别以0、20、40 μmol/L的DAPT作用于HT-29细胞24 h后,采用RT-PCR法检测细胞中的ALDH1、Notch4、DLL1 mRNA,分别采用Western blotting法和免疫组化法检测ALDH1、Notch4、DLL1蛋白.分析HT-29细胞中ALDH1、Notch4、DLL1 mRNA及蛋白表达的相关性.结果 DAPT对HT-29细胞的增殖抑制作用呈一定剂量、时间依赖性,40、80 μmol/L浓度组培养48、72 h时细胞增殖能力低于培养24 h时(P均<0.05).DAPT处理后的细胞形态出现典型凋亡状态改变.20、40 μmol/L的DAPT作用后HT-29细胞中ALDH1、Notch4、DLL1 mRNA相对表达量低于0 μmol/L组(P均<0.05).0、20、40 μmol/L的DAPT作用后HT-29细胞中ALDH1、Notch4、DLL1蛋白相对表达量依次降低(P均<0.05).免疫组化染色图像分析结果示,0、20、40 μmol/L的DAPT作用后HT-29细胞中ALDH1、Notch4、DLL1蛋白表达OD值依次降低(P均<0.05).HT-29细胞中ALDH1、DLL1 mRNA表达与Notch4 mRNA表达均呈正相关关系(r分别为0.997、0.998,P均<0.05).HT-29细胞中ALDH1、DLL1蛋白表达与Notch4蛋白表达均呈正相关关系(r分别为0.998、0.999,P均<0.05).结论 DAPT作用后,细胞增殖受到抑制,细胞中ALDH1、Notch4、DLL1 mRNA和蛋白表达下调;DAPT对HT-29细胞的增殖抑制作用可能与降低肿瘤干细胞特性、下调Notch信号通路受体与配体表达实现的.
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