摘要
目的:观察鱼藤素对MCF-7细胞株细胞增殖和凋亡及磷脂酰肌醇-3激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K/Akt)信号通路的影响,旨在研究其抗肿瘤机制。方法:细胞计数试剂盒8(cell counting kit-8,CCK8)检测0、1、5、10、15和20μmol/L鱼藤素作用6、24、48和72h后对MCF-7细胞增殖的影响,膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染法检测细胞凋亡率,透射电子显微镜观察细胞凋亡形态,蛋白质印迹法检测细胞内PI3K/Akt通路相关分子的蛋白表达。结果:5、10、15和20μmol/L鱼藤素作用6、24、48和72h后能明显抑制MCF-7细胞增殖(P<0.01),抑制率随着浓度升高和时间延长而增加,各组间两两比较差异有统计学意义(P<0.05);而1μmol/L鱼藤素作用6、24、48和72h后对MCF-7细胞的增殖无明显影响(P>0.05)。5、10、15和20μmol/L鱼藤素作用6h后能诱导MCF-7细胞凋亡,透射电子显微镜下可观察到典型的凋亡细胞形态,而相同条件下1μmol/L鱼藤素对MCF-7细胞的凋亡诱导作用不明显。5μmol/L鱼藤素作用6h后,MCF-7细胞内磷酸化Akt(p-Akt)(Thr308)、磷酸化糖原合成酶激酶-3β(phosphorylated glycogen synthase kinase-3β,p-GSK-3β)(Ser9)、磷酸化磷酸肌醇依赖性激酶1(phosphorylated 3-phosphoinositide-dependent protein kinase1,p-PDK1)(Ser241)和磷酸化人第10号染色体缺失的磷酸酶及张力蛋白同源的基因编码的蛋白(phosphorylated phosphatase and tensin homologue deleted on chromosome10,p-PTEN)(Ser380)的表达量明显减少,而总Akt蛋白的表达量则无明显变化;1μmol/L鱼藤素作用6h后,细胞内上述所有蛋白的表达量均无明显变化。结论:鱼藤素可能通过抑制PTEN(Ser380)和PDK1(Ser241)蛋白的磷酸化,进而抑制Akt(Thr308)的磷酸化,间接抑制其下游GSK-3β(Ser9)的磷酸化,最终诱导MCF-7细胞凋亡和抑制其增殖。
Objective：To study the effects of deguelin on proliferation and apoptosis of human breast cancer cell line MCF-7 and on phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway.Methods：After treatment with 0,1,5,10,15 and 20 μmol/L of deguelin for 6,24,48 and 72 hours,the proliferation inhibition rate of MCF-7 cells was measured by cell counting kit-8 assay. Apoptosis rate of MCF-7 cells was detected with Annexin V-fluorescein isothiocyanate/propidium iodide double staining by flow cytometry and the apoptotic morphology was observed under a transmission electron microscope. After treatment with 0,1 and 5 μmol/L of deguelin for 6 hours,5 proteins involved in the PI3K/Akt signaling pathway were examined by Western blot analysis. Results：Deguelin at doses of 5,10,15 and 20 μmol/L inhibited the proliferation of MCF-7 cells at 6,24,48 and 72 hours. There was a significant difference in each group compared with the control group （P0.01）. The inhibitory effect was more marked with increasing concentration and duration of treatment. There were statistical differences （P0.05） among 5,10,15 and 20 μmol/L groups. However,1 μmol/L of deguelin had no obvious effects on the proliferation of MCF-7 cells at 6,24,48 and 72 hours,showing no significant difference compared with control group （P0.05）. Deguelin at doses of 5,10,15 and 20 μmol/L induced apoptosis of MCF-7 cells at 6 hours. There were significant differences （P0.01） in the early and late apoptosis rate between the treated groups and the control group. The typical apoptotic MCF-7 cells were observed under the transmission electron microscopy. However,1 μmol/L of deguelin had no apparent effect in inducing apoptosis of MCF-7 cells at 6 hours. After treatment with 5 μmol/L of deguelin for 6 hours the expression of phosphorylated phosphatase and tensin homologue deleted on chromosome 10 （PTEN） （Ser380）,phosphorylated 3-phosphoinositide-dependent protein kinase 1 （PDK1） （Ser241）,phosphorylated Akt （Thr30
出处
《中西医结合学报》
CAS
2011年第5期533-538,共6页
Journal of Chinese Integrative Medicine
