二萜化合物Rabdocoetsin B抑制蛋白酶体功能并诱导t(8;21)白血病细胞凋亡

Rabdocoetsin B, a diterpenoid isolated from Isodon coetsa, is a potential proteasome inhibitor and induced apoptosis of t(8;21) leukemia cells

摘要:

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1.27 mmol/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、5.0 mmol/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。

Abstract:

Effects of Rabdocoetsin B (Rabd-B), a diterpenoid extracted from Isodon coetsa, on t(8;21) leukemic cells was tested by CCK-8 assay and Flow cytometry. The A549 cells stably expressing pGC-E1-ZU1-GFP were treated with Rabd-B for 4 h, and the accumulation of GFP was detected by fluorescence microscope. Using Western blotting, we investigated the expression of Casp-3, PARP, S6’, which is a subunit of the 19S regulatory complex of the 26S proteasome, and cellular ubiqutinated proteins. We found that Rabd-B induced growth inhibition and apoptosis of Kasumi-1 cells in a dose-dependent manner. In Kasumi-1 cells treated with 2.5 mmol/L Rabd-B for 24 h, pro-caspase-3 was processed into its active form. The substrate of Casp-3, poly ADP-ribose polymerase (PARP), was cleaved with generation of an 85 kD fragment. The increased GFP fluorescence intensity, cleavage of S6’ and the accumulation of ubiquitinated proteins were found in Kasumi-1 cells treated with Rabd-B. These results suggested that Rabd-B is a potential proteasome inhibitor which induces programmed cell death of t(8;21) cells. Further study might provide evidence for employing Rabd-B in treating human t(8;21) leukemia.