摘要
目的 探讨长管贝壳杉素A(longikaurin A,LK-A)对结肠癌细胞的抗肿瘤效应及其潜在的分子机制.方法 将结肠癌HCT116、HT-29细胞分为对照组和实验组,其中对照组给予二甲亚砜溶剂,实验组给予不同浓度LK-A干预,采用CCK-8法评价细胞增殖能力,流式细胞仪分析细胞凋亡情况,以及裸鼠皮下移植瘤实验评价LK-A抑瘤效应,蛋白质印迹法检测PI3K/AKT/mTOR通路蛋白的变化.结果 低浓度的LK-A对结肠癌细胞具有显著生长抑制和促进凋亡的作用.LK-A干预 HCT116 和 HT-29 细胞 24 h 的半抑制浓度(half maximal inhibitory concentration,IC50)分别为 2.18μmol/L 和 1.42μmol/L,48 h 的 IC50 分别为 0.88 μmol/L 和 0.72 μmol/L.在 HCT116 细胞中,2 或 4μmol/L LK-A干预24 h细胞凋亡率分别为(17.36±2.05)％和(34.75±7.01)％,高于对照组(6.60± 1.10)％(P＜0.05);而在 HT-29 细胞中分别为(15.47±1.65)％和(26.30±2.25)％,高于对照组(4.69± 0.91)％(P＜0.05).蛋白质印迹法检测分析显示,随着LK-A浓度增加,磷酸化磷脂酰肌醇-3-激酶(phosphorylated phosphatidylinositol-3 kinase,p-PI3K),磷酸化丝氨酸/苏氨酸蛋白激酶(phosphorylated serine-threonine kinase,p-AKT)和磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin,p-mTOR)表达呈现明显下降趋势.添加AKT激活剂SC79后,LK-A对HCT116和HT-29细胞的增殖抑制和诱导凋亡能力明显下降.裸鼠实验结果提示LK-A具有明显的体内抑瘤作用.结论 LK-A能抑制结肠癌细胞增殖和促进其凋亡发生,其作用机制与抑制PI3K/AKT/mTOR信号通路活化有关.
Abstract
Objective To investigate the antitumor effect of longikaurin A(LK-A)on colon cancer cells and its potential molecular mechanism.Method Colon cancer HCT116 and HT-29 cells were divided into control group and experimental group,and the control group was administered dimethyl sulfoxide solvent,while the experimental group was administered different concentrations of LK-A.The cell proliferation ability was evaluated by CCK-8 assay.The cell apoptosis was analyzed by flow cytometry.The tumor suppressive effect of LK-A was evaluated by subcutaneous xenograft assay in nude mice.The protein changes of PI3K/AKT/mTOR pathway were detected by western blot.Result Low concentrations of LK-A can significantly inhibit the growth of colon cancer cells and increase their apoptosis.The half maximal inhibitory concentration(IC50)of LK-A intervening HCT116 and HT-29 cells for 24 h were 2.18 μmol/L and 1.42 μmol/L,respectively,and the IC50 for 48 h were 0.88 μmol/L and 0.72 µmol/L,respectively.In HCT116 cells,the apoptosis rates of 2 or 4 μmol/L LK-A intervened for 24 h were(17.36±2.05)％and(34.75±7.01)％,which were higher than that of the control group(6.60±1.10)％(P＜0.05);while in HT-29 cells were(15.47±1.65)％and(26.30±2.25)％,higher than that of the control group(4.69±0.91)％(P＜0.05).Western blot analysis showed that with the increase of LK-A concentration,the protein expressions of phosphorylated phosphatidylinositol-3 kinase(p-PI3K),phosphorylated serine-threonine kinase(p-AKT)and phosphorylated mammalian target of rapamycin(p-mTOR)showed a significant downward trend.After adding AKT agonist SC79,the ability of LK-A to inhibit the proliferation and induce apoptosis of HCT116 and HT-29 cells was significantly decreased.The results of experiments on nude mice suggest that LK-A has obvious anti-tumor effect in vivo.Conclusion LK-A inhibits the proliferation of colon cancer cells and increased their apoptosis,and its mechanism is related to the inhibition of PI3K/AKT/mTOR signaling pathway.
维普
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