木香烃内酯和去氢木香内酯对小鼠B16黑色素瘤细胞增殖和凋亡的影响

Study on the effect of costunolide and dehydrocostus lactone on the proliferation and apoptosis of mouse B16 melanoma cells

木香烃内酯(costunolide,CL)和去氢木香内酯(dehydrocostus Lactone,DL)抗恶性黑色素瘤的活性。CL和DL处理B16细胞,采用MTT法检测不同浓度药物对B16细胞增殖的影响;采用DAPI染色荧光显微镜下观察细胞形态变化;Annexin V-FITC/PI双染,流式细胞仪检测细胞凋亡率;Western blot检测凋亡蛋白和PI3K/AKT蛋白表达。结果显示,与对照组比较,CL和DL能显著抑制B16细胞的增殖,随着浓度的增加,抑制率增大。荧光显微镜下观察发现药物处理后细胞形态学发生了改变,出现细胞收缩、高染色质凝聚、凋亡体的可见形成和核降解等细胞凋亡现象。流式细胞术检测确定了能诱导B16细胞凋亡,细胞凋亡率随着药物浓度的增加而增大。Western blot结果显示CL能明显升高Cyt-C、Cleaved caspase-3、Cleaved caspase-9和Bax蛋白的表达,抑制PI3K、AKT和Bcl-2蛋白的表达。CL通过抑制恶性黑色素瘤细胞中PI3K/AKT通路激活,抑制Bcl-2蛋白的表达,促进线粒体膜通透性蛋白Bax的表达,使线粒体膜通透性增加,释放了细胞色素C,从而促进caspase-9和caspase-3的表达,最终促进B16细胞发生凋亡,起到抗黑色素瘤的作用。

To investigate anti malignant melanoma activity induced by costunolide(CL)and dehydrocostus lactone(DL),MTT method was used to detect the inhibitory effect of CL and DL on the proliferation of B16 cells.DAPI staining was used to observe the morphological changes of B16 cells under fluorescence inverted microscope.Annexin V-FITC/PI double staining was used to stain cells and then the apoptosis rates were detected by flow cytometry(FCM).Western blot was used to detect the key protein expression.The results showed that both CL and DL significantly inhibited the proliferation of B16 cells,and the inhibited rates were related with the concentration of CL and DL in does dependent.Under the fluorescence microscope,the morphology of the cells was changed after the treatment with CL or DL,such as cell contraction,high chromatin aggregation,visible formation of apoptotic bodies,et al.FCM results are consistent with the above.The FCM analysis showed that the apoptosis rate was increased after B16 cells treated with CL or DL.The apoptotic rate of B16 cells was increased as CL or DL concentration increased.The results of Western blot showed that the expression of Cyt-C,Cleaved caspase-3,Cleaved caspase-9 and Bax were increased in CL group compared with control group,while the expression of PI3K,AKT and Bcl-2 proteins were decreased.In conclusion,CL exhibited anti-melanoma activity,and it may be realized by inhibiting the activation of PI3K/Akt pathway,inhibiting the expression of Bcl-2 protein,increasing the mitochondrial membrane permeability protein Bax,releasing Cyt-C,and increasing the expression of caspase-9 and caspase-3.