苦参碱诱导H466肺癌细胞凋亡及对Wnt信号通路的影响

Matrine inducing apoptosis of H466 lung cancer cells and its effect on Wnt signaling pathway

目的探讨苦参碱诱导H466肺癌细胞凋亡及对Wnt信号通路的影响。方法将肺癌H466细胞分为三组,对照组为H466细胞,低处理组为H466细胞+0.5g的苦参碱;高处理组为H466细胞+2.0g的苦参碱,采用MTT、免疫双荧光、流式细胞仪、免疫印迹及qRT-PCR检测肺癌H466细胞的凋亡率、细胞周期及Wnt的蛋白表达及β-cateninmRNA表达。结果MTT实验证明,低处理组的肺癌H466细胞活性低于对照组(P<0.05),高处理组的肺癌H466细胞活性低于低处理组(P<0.05),说明苦参碱对肺癌H466细胞具有抗肿瘤作用;对照组为正常的肺癌H466细胞呈蓝色荧光表达的活细胞,低处理组发现H466细胞呈现早期凋亡并细胞核破碎形成凋亡小体,凋亡程度高于对照组(P<0.05),死亡的肺癌细胞呈现荧红色,在高处理组中的死亡率高于低处理组(P<0.05);H466细胞在苦参碱作用下,在G0/G1期细胞比例升高,S期呈现下降趋势,对照组与低处理组相比差异较小(P>0.05),高处理组与低处理组比较有差异(P<0.05),但在G2/M无明显改变,G0/G1在高处理组凋亡率呈现增高的趋势;低处理组中的Wnt-2信号通路中的表达少于对照组(P<0.05),与低处理组相比高处理组中表达最低(P<0.05);qRT-PCR显示:低处理组的β-catenin mRNA表达少于对照组,在高处理组中β-catenin mRNA表达少于低处理组。结论苦参碱可以加快肺癌H466细胞凋亡,在这过程中可能与阻断Wnt信号通路传导有关,苦参碱可以阻断Wnt信号表达,抑制肺癌细胞分化。

Objective To investigate the effect of matrine on apoptosis and Hnt signaling pathway in H466 lung cancer cells.Methods H466 cells of lung cancer were divided into three groups.The control group was H466 cells,the low-treatment group was H466 cells+0.5 g of matrine,and the high-treatment group was H466 cells+2.0 g of matrine.It used MTT,immune doubles,fluorescence,flow cytometry,immunoblotting and qRT-PCR to detect the apoptosis rate,cell cycle,Wnt protein expression andβ-catenin mRNA expression of lung cancer H466 cells.Results MTT experiment showed that the concentration of matrine increased,and the inhibitory activity of lung cancer H466 cells increased.Matrine had anti-tumor effect on lung cancer H466 cells.The control group was a normal lung cancer H466 cells with blue fluorescent expression of live cells,and the low-treatment group found that H466 cells exhibited early apoptosis and nuclear fragmentation to form apoptotic bodies with higher apoptosis. In the control group, the high-treatment group had the highest mortality ( P <0.05). The ratio of H466 cells in G0/G1 phase increased under the action of matrine. The S phase showed a downward trend, and the difference between the two groups was relatively large ( P >0.05). But there was no significant change in G2/M and G0/G1. The apoptosis rate of the high-treatment group showed an increasing trend. The immunoblotting showed that the expression of Wnt signaling pathway protein was lower in the low-treatment group than in the control group ( P <0.05). The expression of Wnt signaling pathway protein was the lowest in the high processing group( P <0.05). qRT-PCR showed that the expression of β-catenin mRNA in the high-treatment group was the least compared with the other two groups, and the expression was the most active in the control group, followed by the low expression group. Conclusion Matrine can accelerate the apoptosis of H466 cells in lung cancer, which may be related to the blockade of Wnt signaling pathway. Matrine can block the expression of Wnt signaling and inhibit the differentiation of lung cancer cells.