金复康口服液诱导肺癌细胞A549凋亡的机制

Mechanism of Jinfukang Oral Liquid in Inducing Apoptosis of Non-small Cell Lung Cancer Cell Line A549

目的：研究金复康口服液（JFK）诱导人非小细胞肺癌细胞A549凋亡的作用机制。方法：取对数生长期的人非小细胞肺癌细胞A549,随机分为空白组和药物组,采用溴化四甲基偶氮（MTT）比色法,考察不同质量浓度JFK（0.3,1.5,3,7.5,15,30 g·L（-1））对A549细胞体外增殖能力的影响。通过平板克隆形成实验考察JFK对A549细胞平板克隆形成的影响,利用流式细胞仪考察JFK对A549细胞凋亡的影响;蛋白免疫印迹法（Western blot）考察JFK对裂解DNA修复酶（cleaved PARP）,活化型半胱天冬酶-3（actived Caspase-3）,Wnt/β-联蛋白（β-catenin）,细胞周期蛋白D1（cyclin D1）的蛋白表达及对促分裂原活化蛋白激酶（MAPK）信号通路和蛋白激酶B（Akt）信号通路的影响,实验中设置空白组和药物组,药物组为A549细胞加入15,30 g·L（-1）JFK,分别干预24 h,30 min。结果：JFK在1.5-30 g·L（-1）呈质量浓度依赖性的抑制A549细胞外增殖（P〈0.01）。JFK抑制A549细胞的平板克隆形成在15-30 g·L（-1）,其中JFK 30 g·L（-1）时抑制A549细胞的平板克隆最显著（P〈0.01）。与空白组比较,JFK 15-30 g·L（-1）可提高AnnexinⅤ和PI双阳细胞率,诱导A549细胞凋亡（P〈0.05）,且呈浓度依赖性提高cleaved PARP和actived Caspase-3的蛋白表达,并抑制β-catenin和cyclin D1在蛋白水平的表达（P〈0.05,P〈0.01）。JFK上调MAPK信号通路中JNK和p38的蛋白磷酸化水平（P〈0.05）,同时下调Akt信号通路中Akt在T308和S473的位点磷酸化水平（P〈0.05）。结论：金复康口服液抑制人非小细胞肺癌细胞A549的体外增殖、平板克隆的形成并诱导其凋亡,部分通过调控JNK/p38/MAPK信号通路和Akt信号通路,抑制β-catenin和cyclin D1,同时提高凋亡相关蛋白cleaved PARP和actived Caspase-3的表达。

Objective：To explore the mechanism of Jinfukang oral liquid（JFK） in inducing the apoptosis of non-small cell lung cancer cell line A549.Method：The non-small cell lung cancer cell lines A549 in logarithmic phase of growth were randomly divided into the control group and drug treatment groups.The 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay was used to investigate the effects of different concentrations of JFK（0.3,1.5,3,7.5,15,30 g·L^（-1）） on the proliferation of A549 cells in vitro.The plate colony formation assay and flow cytometry were used to observe the effects of JFK on the colony formation and apoptosis of A549 cells,respectively.Protein expressions of cleaved PARP,active Caspase-3,β-catenin,cyclin D1,and the mitogen-activated protein kinase（MAPK）/proteinkinse B（Akt） signaling pathways were investigated by Western blot.Cells were divided into control group and drug groups treated at the concentrations of15,30 g·L^（-1）,and each group was intervened for 24 h or 30 min.Result：JFK inhibited the proliferation of A549 cells in a dose-dependent manner within the range from 1.5-30 g·L^（-1）（P 〈 0.01）.JFK inhibited colony formation at 15-30 g·L^（-1）（P 〈 0.01）.JFK increased the ratio of Annexin V and PI double positive cells at the concentrations of 15-30 g·L^（-1）,suggesting that JFK induced apoptosis in A549 cells（P 〈 0.05）.The protein expressions of cleaved PARP and activated Caspase-3 were increased,while the expressions of β-catenin and cyclin D1 were suppressed in a dose-dependent manner（P 〈 0.05,P 〈 0.01） after treatment with JFK.And the phosphorylation levels of JNK and p38 were up-regulated,while that of T308 and S473 sites of Akt were downregulated（P 〈 0.05）.Conclusion：Jinfukang decoction inhibited the proliferation and colony formation of nonsmall cell lung cancer cells A549.JFK induced apoptosis with the increase of the protein expressions of cleaved PARP and activated Caspase-3,and the decrease of the protein expressions of β-catenin and cyclin D1,partly through JNK/p38/MAPK and Akt signaling pathways.