Notch3信号通路介导 SAHA 诱导的小细胞肺癌H446细胞凋亡

摘要

目的：观察辛二酰苯胺异羟肟酸（suberoylanilide hydroxamic acid， SAHA）对人小细胞肺癌 H446细胞凋亡的影响，并探讨其分子机制。方法：选取人小细胞肺癌 H446细胞作为研究对象，采用 CCK－8法检测SAHA的细胞毒作用并测定 IC50，采用流式细胞术检测细胞凋亡，转染 N3ICD 真核表达质粒构建高表达 N3ICD 的 H446细胞系，采用 RT－PCR 法检测 Notch3的 mRNA 水平，采用 Western blot 法检测 Notch3、N3ICD、Puma 和 cleaved caspase－3的蛋白水平。结果： SAHA 可显著降低 H446细胞存活率且呈剂量依赖性（P ＜0．05），SAHA 作用48 h 的 IC50为1．91μmol／L；SAHA 可诱导 H446细胞凋亡且具有剂量依赖性（P ＜0．05）；H446细胞中 Notch3基因表达呈阴性， SAHA可使 H446细胞 Notch3基因恢复表达并激活 Notch3信号通路（P ＜0．05）；沉默 Notch3基因可抑制 SAHA 对H446细胞的促凋亡作用（P ＜0．05）；N3ICD 高表达使 H446细胞中 Puma 和 cleaved caspase－3蛋白水平升高（P ＜0．01）。结论：在体外 SAHA 可激活人小细胞肺癌 H446细胞 Notch3信号通路，上调 Puma 蛋白表达水平，诱导H446细胞凋亡。%AIM: To investigate the effect of suberoylanilide hydroxamic acid (SAHA) on the apoptosis of hu-man small-cell lung cancer H446 cells and its possible mechanism.METHODS: H446 cells were incubated in the medi-um containing SAHA.CCK-8 assay was used to detect the anti-tumor effect of SAHA on the H446 cells, and IC50 values of SAHA were calculated.Flow cytometry was used to analyze the apoptosis.After Notch3 gene was silenced, the pro-apopto-tic effect of SAHA on the H446 cells was inhibited ( P <0.05).Eukaryotic expression plasmid containing N3ICD was transfected into the H446 cells, so that N3ICD was expressed in the H446 cells.The mRNA expression of Notch3 was measured by RT-PCR.The protein levels of Notch3, N3ICD, Puma and cleaved caspase-3 were determined by Western blot.RESULTS: SAHA remarkably reduced the cell viability in a dose-dependent manner (P <0.05), and the IC50 value of SAHA was 1.91 μmol/L.SAHA induced apoptosis in a dose-dependent manner (P <0.05).The expression of Notch3 gene was negative in the H446 cells, SAHA reactivated Notch3 gene and Notch3 pathway in a dose-dependent manner (P <0.05).Notch3 knockdown inhibited apoptosis induced by SAHA (P <0.05).Over-expression of N3ICD up-regula-ted the protein levels of Puma and cleaved caspase-3.CONCLUSION: SAHA induces apoptosis in human small-cell lung cancer H446 cells by activating Notch3 pathway and up-regulating the protein level of Puma.