摘要
目的研究龙葵碱对人乳腺癌MCF-7细胞凋亡的影响并探讨相关机制。方法采用四甲基偶氮唑蓝(MTT)法观察不同浓度龙葵碱对人乳腺癌MCF-7细胞的生长抑制作用;4,6-二脒基-2-苯基吲哚(DAPI)染色荧光显微镜进行细胞凋亡核形态学观察;DNA琼脂糖凝胶电泳进行DNA片段化分析,FITC-Annexin V/PI荧光标记流式细胞术检测细胞凋亡率,荧光显微镜结合Fluo-8/Am法、流式细胞术和分光光度比色法分别检测胞内Ca2+浓度、线粒体膜电位(△ψm)和caspase-3,caspase-8活性变化。结果四甲基偶氮唑蓝法结果显示,龙葵碱对人乳腺癌MCF-7细胞有生长抑制作用。10.0 mmol·L-1龙葵碱处理2d,4,6-二脒基-2-苯基吲哚染色可见核浓缩及边缘现象,DNA电泳出现特征性的凋亡条带。10.0 mmol·L-1龙葵碱处理1,2,3d的细胞凋亡率分别为(20.9±7.3)%、(42.6±8.8)%和(74.9±12.8)%;细胞内Ca2+荧光强度分别为35.6±2.9、52.3±5.6和27.2±2.2;线粒体膜电位(△ψm)值分别下降7.7%、33.2%和46.9%;caspase-8活性在1 d达最高(1.85±0.09)U·μg-1,caspase-3活性则在2 d达最高(2.18±0.09)U·μg-1,与对照组相比均有统计学显著性差异(P<0.05)。结论龙葵碱可诱导人乳腺癌MCF-7细胞凋亡,其诱导凋亡的机制可能与胞内Ca2+浓度升高、线粒体膜电位降低和caspase-3、8活化有关。
OBJECTIVE To study the effects of solanine on apoptosis of MCF-7 cells and explore the related mechanism. METHODS MTT assay was used to test the inhibitory effect on MCF-7 cellular proliferation with varying concentrations of solanine. The morphological changes of MCF-7 cells were observed with fluorescence microscope after DAPI staining. The analysis of DNA frag- mentation on MCF-7 cells was tested by DNA agarose gel eleetrophoresis. The apoptosis rates and alternation of mitochondrial mem- brane potential （AOm） in MCF-7 cells were measured by flow cytometry using fluorescein labeling FITC-Annexin V/PI and JC-1. In- tracellular Ca2. was determined by fluorescence microscope. The activities of caspase-3 and -8 in apoptosis were detected by eolorimet- fie assay. RESULTS The MTT assay showed that solanine inhibited MCF-7 cells proliferation. After 10. 0 mmol · L^-l solanine treat- ment for 2 day, the phenomenon of nuclear condensation and marginalization were shown by DAPI staining in MCF-7 cells, and there was characteristic DNA bands of apoptosis detected by the DNA agarose gel electrophoresis. The apoptosis rate were （ 20. 9 ± 7.3 ） %, （42. 6±8. 8）% and （74. 9 ± 12. 8）% at 1, 2 and 3 d treatment, respectively. The fluorescence intensity of intraeellular Ca^2＋ were 35.6±2. 9, 52. 3 ± 5.6 and 27.2 ± 2. 2 at the same time. The alternation of mitoehondrial membrane （ Aψm） was decreased to 7.7% , 33.2% and 46. 9% compared to control. The maximal activities of intracellular caspase-8 was （ 1.85±0. 09） U · μg^-1 after 1 d sola- nine treatment while the easpase-8 activity reached the highest point after 2 d treatment which was （2. 18±0. 09） U · μg^-1, respec- tively. There were significantly difference compared with the control groups （ P 〈 0. 05 ）. CONCLUSION Solanine can inhibit MCF- 7 proliferation and induce apoptosis. The molecular mechanism may be related to the increased concentration of intracellular Ca^2＋ , the decrease of mitochondrial membrane potential and
出处
《中国药学杂志》
CAS
CSCD
北大核心
2014年第16期1404-1409,共6页
Chinese Pharmaceutical Journal
基金
嘉兴学院实验技术专项(70113004BW)
