Notch3信号通路介导SAHA诱导的小细胞肺癌H446细胞凋亡  

Notch3 pathway mediates SAHA-induced apoptosis in human small-cell lung cancer H446 cells

作　　者：陈红莲[1] 刘辉[1] 杨旭光[1] 袁磊[1] 

机构地区：[1]漯河医学高等专科学校,河南漯河462002

出　　处：《中国病理生理杂志》2016年第9期1556-1561,共6页Chinese Journal of Pathophysiology

基　　金：河南省科技厅科技发展计划项目(No.142102310203);漯河医学高等专科学校自然科学研究计划项目(No.2013-S-LMC04)

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 human small-cell lung cancer H446 cells and its possible mechanism. METHODS： H446 cells were incubated in the medium 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-apoptotic effect of SAHA on the H446 cells was inhibited（ P〈0. 05）. Eukaryotic expression plasmid containing N3 ICD was transfected into the H446 cells,so that N3 ICD was expressed in the H446 cells. The mRNA expression of Notch3 was measured by RT-PCR. The protein levels of Notch3,N3 ICD,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 N3 ICD up-regulated 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.

关 键 词：辛二酰苯胺异羟肟酸 小细胞肺癌 NOTCH3 细胞凋亡 H446细胞 

分 类 号：R734.2[医药卫生—肿瘤] R730.23[医药卫生—临床医学]