摘要
目的探讨PI3K/AKT通路在吴茱萸碱(evodiamine,EVO)诱导小细胞肺癌H1688和H446细胞凋亡中的作用。方法分别以不同浓度(1、5、20μmol/L)和不同作用时间(3、6、12、24、48 h)的EVO处理H1688和H446细胞,Western blot检测p-AKT的表达水平。以EVO分别作用于经过PI3K/AKT通路激活剂IGF-1或抑制剂LY294002预处理后的H1688和H446细胞,Western blot检测p-AKT蛋白的表达。将EVO作用于IGF-1预处理的H1688细胞后,Annexin-V/PI染色检测细胞凋亡率,Western blot检测Bcl-2、Bax、Caspase-3蛋白的表达水平。以不同浓度的EVO处理H1688细胞,Western blot检测t-AKT、PTEN蛋白表达,RT-PCR检测AKT m RNA表达。结果与对照组相比,不同浓度和不同作用时间的EVO处理后H1688和H446细胞中p-AKT表达水平下降(P<0.05)。而IGF-1可减弱EVO下调H1688和H446细胞p-AKT的作用(P<0.05),并降低EVO在H1688中的增殖抑制和凋亡诱导作用。EVO与LY294002下调p-AKT蛋白的效果差异无统计学意义(P>0.05)。EVO对H1688细胞t-AKT蛋白和AKT m RNA的表达无影响,EVO可明显上调PTEN的表达(P<0.05)。结论抑制PI3K/AKT通路活性是EVO诱导H1688和H446细胞凋亡的重要机制之一。
Objective To study the role of evodiamine （EVO） in inducing apoptosis of small-cell lung cancer H1688 and H446 cells by inhibiting PI3K/AKT pathway. Methods H1688 and H446 cells were treated with EVO at different concentrations （1, 5, 20 ixmol/L） for different time periods （3, 6, 12, 24 and 48 h）, respectively. Thereafter, p-AKT was assayed by Western blotting. After pretreatment with PI3K/AKT pathway activator IGF-1 or inhibitor LY294002, H1688 and H446 cells were treated with EVO. Subsequently, p-AKT was detected by Western blotting. After H1688 cells were pretreated with IGF-1 and then treated with EVO, they were stained with Annexin-V/PI, and analyzed by flow cytometry. The expression of Bcl-2, Bax and Caspase-3 was detected by Western blotting respectively. After H1688 cells were treated with different concentrations of EVO, the expression levels of PTEN and t-AKT were assayed byWestern blotting, and AKT mRNA level was detected by RT-PCR. Results Compared with the control group, p-AKT expression was decreased in H1688 and H446 cells treated with different concentrations of EVO for different times （P 〈 0.05）. In addition, IGF-1 attenuated down-regulation of p-AKT in H1688 and H446 cells induced bv EVO （ P 〈 0. 05 ） , and inhibited the apoptosis of H1688 cells induced by EVO. There was no significant difference between EVO and LY294002 in the effect of down-regulating p-AKT （ P 〉 0.05 ）. EVO showed no significant effect on the expression of t-AKT protein and AKT mRNA （P 〉 0. 05 ）. However, EVO significantly increased the expression of PTEN protein（P 〈 0. 05 ）. Conclusion Inhibition of PI3K/AKT pathway plays a key role in EVO-induced apoptosis of small-cell lung cancer H1688 and H446 ceils.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2016年第4期330-337,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81172239)~~
