作者：李悦予，冯霞

单位：南京医科大学附属脑科医院药学部，江苏 南京 210029

Authors: Li Yueyu, Feng Xia

Unit:  Department of Pharmacy, Brain Hospital Affiliated to Nanjing Medical University, Nanjing 210029, Jiangsu, China

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2.18 μmol/L 和1.42 μmol/L，48 h的IC50分别为0.88 μmol/L 和0.72 μmol/L。在HCT116细胞中，2 或4 μmol/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（phosphorylatedserine-threonine kinase, p-AKT）和磷酸化哺乳动物雷帕霉素靶蛋白（phosphorylated mammalian target ofrapamycin, 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。

关键词： 长管贝壳杉素A；结肠癌；增殖；凋亡

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.

Key Words:  Longikaurin A; Colon cancer; Proliferation; Apoptosis

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