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从香茶菜属植物细锥香茶菜中分离得到的二萜类化合物Rabdocoetsin B(Rabd-B),研究其对人类t(8;21)白血病细胞凋亡的诱导作用及其对蛋白酶体19S泛素识别亚基S6’(Rpt5)的抑制作用,为Rabd-B应用于t(8;21)白血病治疗提供实验依据。以不同浓度的Rabd-B处理t(8;21)白血病Kasumi-1细胞,应用CCK-8法检测细胞活力,用Bliss法计算IC50值,通过流式细胞术检测细胞凋亡情况,并以稳定表达pGC-E1-ZU1-GFP的A549细胞作为蛋白酶体抑制剂筛选模型,荧光显微镜成像拍照GFP荧光。Western blotting检测不同浓度的Rabd-B处理Kasumi-1,检测Casp-3、S6’(Rpt5)、PARP、ubiqutin在该细胞内的表达变化。结果显示,Rabd-B明显抑制t(8;21)阳性的Kasumi-1细胞生长,48 h的IC50值为1.27μmol/L;同时诱导Kasumi-1细胞凋亡效果显著;荧光显微镜观察药物处理和未处理的pGC-E1-ZU1-GFP的A549细胞Ub-GFP表达,结果显示GFP平均荧光值随药物浓度增高而增强;Rabd-B处理Kasumi-1细胞24、48 h后蛋白质免疫印迹技术检测Casp-3、PARP,发现Casp-3激活,其底物PARP发生切割产生85 kD的降解带;2.5、5.0μmol/L Rabd-B处理Kasumi-1细胞24、48 h后Western blotting检测蛋白酶酶体组分,发现19S调节亚基S6’(Rpt5)降解,细胞内泛素化水平增高。以上结果说明,Rabd-B一方面通过激活Caspase级联反应,另一方面抑制蛋白酶体19S泛素识别亚基,导致细胞内泛素化积累,进而抑制t(8;21)白血病细胞生长并诱导其凋亡。
Rabdocoetsin B (Rabd-B), a diterpenoid isolated from the genus Chaxingu, was used to investigate its effect on the apoptosis of human leukemia cells and its effect on proteasome 19S Ubiquitin recognition subunit S6 ’(Rpt5) inhibition, Rabd-B for the application of t (8; 21) to provide experimental evidence of the treatment of leukemia. Kasumi-1 cells were treated with different concentrations of Rabd-B and the cell viability was determined by CCK-8. The IC50 value was calculated by Bliss method. The cell apoptosis was detected by flow cytometry, A549 cells expressing pGC-E1-ZU1-GFP served as a proteasome inhibitor screening model and fluorescence microscopy imaging of GFP fluorescence. Western blotting was used to detect the expression of Casp-3, S6 ’(Rpt5), PARP and ubiqutin in Rabd-B cells treated with different concentrations of Kasumi-1. The results showed that Rabd-B significantly inhibited the growth of t (8; 21) -positive Kasumi-1 cells at 48 h with an IC50 value of 1.27 μmol / L and significant apoptosis effect on Kasumi-1 cells. Fluorescence microscopy showed that the drug- Ub-GFP expression of untreated pGC-E1-ZU1-GFP A549 cells showed that the average fluorescence value of GFP increased with the increase of drug concentration; Kasumi-1 cells were treated with Rabd-B for 24,48 h after Western blotting to detect Casp -3, PARP. Casp-3 was found to activate and its substrate PARP cleaved to produce a 85 kD degradation band. Kasumi-1 cells were treated with 2.5, 5.0μmol / L Rabd-B for 24,48 h. Western blotting was used to detect proteasome The 19S regulatory subunit S6 ’(Rpt5) was found to be degraded and the intracellular ubiquitination level was increased. The above results indicate that Rabd-B inhibits the growth of t (8; 21) leukemic cells and induces, on the one hand, activation of the caspase cascade and, on the other hand, inhibition of the proteasomal 19S ubiquitin recognition subunit, resulting in ubiquitination of cells Its apoptosis.