白花丹醌增强TRAIL诱导Kasumi-1细胞凋亡及其机制

Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells

目的:观察白花丹醌、rsTRAIL单独及其联合体外诱导Kasumi-1细胞凋亡的作用,探讨其作用机制。方法:采用WST-8(CCK-8)比色法测定rsTRAIL、白花丹醌单独和联合应用对Kasumi-1细胞的生长抑制率;用流式细胞术、TUNEL法观察并且检测凋亡率;实时定量PCR检测白花丹醌作用后DR4和DR5 mRNA水平变化;Western blotting法检测单独应用及联合应用后DR5、caspase-3、caspase-8、caspase-9、Bid、Bax及c-FLIP的变化。结果:(1)白花丹醌和rsTRAIL单独应用均可抑制Kasumi-1细胞的生长,联合应用可增加对Kasumi-1细胞的生长抑制率且呈时间和剂量依赖性(P<0.05)。(2)rsTRAIL(100μg/L)和白花丹醌(2μmol/L)单用及其联合使用诱导Kasumi-1细胞Annexin V阳性细胞百分率分别为(27.7±2.9)%、(25.6±3.1)%和(52.1±3.3)%。(3)TUNEL法检测发现联合应用组较单用组凋亡细胞显著增多。(4)实时定量PCR检测发现白花丹醌可以上调DR5的表达。(5)Western blotting发现白花丹醌单独作用及其联合rsTRAIL应用时上调DR5、激活caspase-8和下调c-FLIP表达。结论:白花丹醌具有增强TRAIL诱导Kasumi-1细胞凋亡的作用,其机制与DR5上调、caspase-8激活和c-FLIP下调有关。

AIM： To investigate the effect of plumbagin and tumor necrosis factor-related apoptosis-inducing ligand（TRAIL） on the apoptosis of leukemic Kasumi-1 cells. METHODS： Kasumi-1 cells were treated with plumbagin alone,recombinant soluble TRAIL（rsTRAIL） alone or the combination of plumbagin with rsTRAIL to induce apoptosis.The cell proliferation was analyzed by CCK-8 assay.Apoptosis was determined by flow cytometry with AnnexinⅤ/PI double staining and TUNEL staining.The expression of DR4 and DR5 at mRNA level was measured by real-time PCR.The expression of signal transduction proteins,such as DR5,caspase-3,caspase-8,caspasep-9,Bid,Bax and c-FLIP was detected by Western blotting. RESULTS： Both rsTRAIL and plumbagin induced the apoptosis in Kasumi-1 cells,and combination of plumbagin with rsTRAIL enhanced the apoptosis.The ratios of Annexin V-positive Kasumi-1 cells were（27.7±2.9）%,（25.6±3.1）% and（52.1±3.3）% in 100 μg/L rsTRAIL group,2 μmol/L plumbagin group and the combination group,respectively,and the positive rate in combination group was significantly higher than those in other 2 groups.TUNEL assay demonstrated that the number of apoptotic cells in combination group was higher than that in the cells treated with rsTRAIL or plumbagin alone.Plumbagin up-regulated the expression of DR5 at mRNA level in Kasumi-1 cells,and up-regulation of DR5,activation of caspase-8 and down-regulation of c-FLIP at protein level were detected in the cells treated with plumbagin alone and the combination of plumbagin with rsTRAIL. CONCLUSION： Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells by up-regulating DR5,activating caspase-8 and down-regulating c-FLIP.