摘要
目的 探讨吴茱萸碱(EVO)在人肺癌NCI-H1299细胞中的抗肿瘤作用及其可能机制.方法 应用CCK-8 法检测 EVO(1.0、2.5、5.0、7.5、10.0、12.5、15.0 μmol/L)处理 24、48、72 h 对 NCI-H1299 细胞活力的影响,采用PI单染流式法和Annexin V-FITC+PI双染流式法分别检测EVO对NCI-H1299细胞周期和细胞凋亡的影响,Western blot法检测相关蛋白表达.结果 与二甲基亚砜(DMSO)对照组相比,EVO能显著抑制NCI-H1299细胞的增殖活性,细胞增殖活力随着EVO浓度增加呈下降趋势,不同给药浓度组间比较差异有显著性(F=5 091.83,P＜0.01),不同时间点间比较差异有显著性(F=1 241.80,P＜0.01),且细胞增殖活力变化呈浓度和时间依赖性.12.5 μmol/L EVO处理细胞24 h后,与DMSO对照组相比,NCI-H1299被阻滞于G2/M期,NCI-H1299细胞凋亡率显著上升(t=35.52,P＜0.001),细胞周期相关蛋白Cdc2磷酸化水平及P53、CyclinB1、Cdc25C(间期)蛋白水平明显上升(F=13.55～69.14,P＜0.05),细胞凋亡相关蛋白胱天蛋白酶-3(caspase-3)、胱天蛋白酶-9(caspase-9)和多聚腺苷二磷酸核糖聚合酶1(PARP1)表达增高(F=21.62～196.54,P＜0.05),B细胞淋巴瘤因子-2(Bcl-2)/Bcl-2相关X蛋白(Bax)比值明显降低(F=3.86,P＜0.05),受体相互作用蛋白1(RIP1)、受体相互作用蛋白3(RIP3)和混合系激酶区域样蛋白(MLKL)磷酸化水平明显上升,差异均有统计学意义(F=71.69～118.33,P＜0.05).结论 EVO可在NCI-H1299细胞中诱导细胞增殖抑制、G2/M细胞周期阻滞、内源性凋亡和程序性坏死,从而发挥抗肿瘤作用.
Abstract
Objective To investigate the antitumor effect of evodiamine(EVO)on human lung cancer NCI-H1299 cells and its possible mechanism.Methods CCK-8 assay was used to observe the effect of EVO(1.0,2.5,5.0,7.5,10.0,12.5,and 15.0 pmol/L)on the viability of NCI-H1299 cells after 24,48,and 72 h of treatment;PI staining and Annexin V-FITC+PI double staining were used to observe the effect of EVO on the cell cycle and apoptosis of NCI-H1299 cells;Western blot was used to mea-sure the expression of proteins.Results Compared with the dimethyl sulfoxide(DMSO)control group,EVO significantly inhi-bited the proliferative activity of NCI-H1299 cells,and cell proliferative activity tended to decrease with the increase in EVO con-centration;there was a significant difference between different concentration groups(F=5 091.83,P＜0.01)and between different time points(F=1 241.80,P＜0.01),and cell proliferative activity changed in a concentration-and time-dependent manner.After the cells were treated with 12.5 μmol/L EVO for 24 h,NCI-H1299 cells were arrested in G2/M phase compared with the DMSO control group,and there were significant increases in the apoptosis rate of NCI-H1299 cells(t=35.52,P＜0.001),the phosphory-lation level of the cell cycle-related protein Cdc2,and the levels of P53,CyclinB1,and Cdc25C(mitotic phase)proteins(F=13.55-69.14,P＜0.05).There were also significant increases in the expression levels of the cell apoptosis-related proteins caspase-3,caspase-9,and PARP1(F=21.62-196.54,P＜0.05),a significant reduction in B-cell lymphoma factor-2/Bcl-2-associated X-protein ratio(F=3.86,P＜0.05),and significant increases in the phosphorylation level of receptor-interacting protein 1,recep-tor-interacting protein 3,and mixed-lineage kinase domain-like protein(F=71.69-118.33,P＜0.05).Conclusion EVO can in-duce the inhibition of cell proliferation,G2/M cell cycle arrest,endogenous apoptosis,and programmed death of NCI-H1299 cells,thereby exerting an antitumor effect.
维普
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